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Immunolocalization of matrix proteins in nacre lamellae and their in vivo effects on aragonitic tablet growth
Journal of Structural Biology 164 (2008) 33–40
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Immunolocalization of matrix proteins in nacre lamellae and their in vivo effects on aragonitic tablet growth Ningping Gong a, Junlong Shangguan a, Xiaojun Liu a, Zhenguang Yan a, Zhuojun Ma a, Liping Xie a,b,*, Rongqing Zhang a,b,* a b
Institute of Marine Biotechnology, Department of Biological Sciences and Biotechnology, Tsinghua University, Beijing 100084, China Protein Science Laboratory of the Ministry of Education, Tsinghua University, Beijing 100084, China
a r t i c l e
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Article history: Received 9 January 2008 Received in revised form 23 May 2008 Accepted 24 May 2008 Available online 3 June 2008 Keywords: Mollusk shell nacre Matrix protein BMP-2 Nacrein Antibodies
a b s t r a c t How matrix proteins precisely control the growth of nacre lamellae is an open question in biomineralization research. Using the antibodies against matrix proteins for immunolabeling and in vivo experiments, we investigate the structural and functional roles of EDTA–soluble matrix (SM) and EDTA– insoluble matrix (ISM) proteins in nacre biomineralization of the pearl oyster Pinctada fucata. Immunolabeling reveals that a SM protein, nacrein, distributes within aragonitic tablets and intertabular matrix. An ISM protein, which we named P43, has been specifically recognized by polyclonal antibodies raised against the recombinant protein of P. fucata bone morphogenetic protein 2 in immunoblot analysis. Immunolabeling indicates that P43 is localized to interlamellar sheet, and also embedded within aragonitic tablets. Although nacrein and P43 both distribute within aragonitic tablets, they function differently in aragonitic tablet growth. When nacrein is suppressed by the antibodies against it in vivo, crystal overgrowth occurs, indicating that this SM protein is a negative regulator in aragonitic tablet growth. When P43 is suppressed in vivo, the organo-mineral assemblage is disrupted, suggesting that P43 is a framework matrix. Taken together, SM and ISM proteins are indispensable factors for the growth of nacre lamellae, controlling crystal growth and constructing the framework of aragonitic tablets. Ó 2008 Elsevier Inc. All rights reserved.
1. Introduction Nacre (mother-of-pearl) is a biogenetic material widely distributing in mollusks, and usually forms in the inner layer of shells. The structure of nacre from bivalves is characterized as an arrangement of continuous parallel lamellae (Fig. 1A), separated by sheets of interlamellar matrix (Wada, 1968; Addadi et al., 2006). Each lamella is composed of polygonal aragonitic tablets (Fig. 1B), which is sealed to each other by intertabular matrix. An aragonitic tablet seems to be a single crystal, but actually is a coherent aggregation of crystalline nanograins (about 45 nm mean size) with the same crystallographic orientation, connected by a continuous organic framework (Rousseau et al., 2005a; Stolarski and Mazur, 2005). Nacre is an organo-mineral assemblage with dominant calcium carbonate and a minor organic matrix complex, resulting in superior mechanical properties and excellent osteoinductive activity (Silve et al., 1992; Lamghari et al., 1999; Weiner et al., 2003). The organic matrix complex includes proteins and polysaccharides, which is thought to direct the growth of calcium carbonate crystal * Corresponding authors. Fax: +86 10 62772899. E-mail addresses: [email protected] (L. Xie), [email protected] (R. Zhang). 1047-8477/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.jsb.2008.05.009
and be responsible for the extraordinary properties of nacre (Weiner and Addadi, 1997; Mouriès et al., 2002; Zhang and Zhang, 2006). Matrix proteins in nacre are usually classified into two groups depending on their solubility in decalcifying solution (Crenshaw and Ristedt, 1976). EDTA-based solution is often used for decalcification. EDTA–soluble matrix (SM) proteins have been proposed to localize within calcium carbonate crystals as intracrystalline matrix (Crenshaw, 1972) and control crystal morphology, crystal phase switching and orientation (Mann et al., 1993; Belcher et al., 1996; Zhang and Zhang, 2006). In contrast, EDTA–insoluble matrix proteins (ISMs) are hypothesized to localize around calcium carbonate crystallites and thus are supposed as intercrystalline matrices and framework macromolecules (Crenshaw, 1972; Sudo et al., 1997). Matrix proteins play vital roles in the formation of nacre. But how matrix proteins interact with the mineral in the self-organization of nacre has not been fully understood yet. Immunolocalization has been widely used to study the functions of matrix proteins in the growth of biomaterials, such as sea urchin, coral skeletons and shell (Crenshaw and Ristedt, 1976; Cho et al., 1996; Puverel et al., 2005; Nudelman et al., 2006; Marin et al., 2007). Antibody inhibition assay is proved to be a reliable method for in vivo investigation of the functions of matrix proteins in the growth of nacre lamellae (Ma et al., 2007).
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Fig. 1. Nacre structure of P. fucata. (A) SEM view shows the cross-section of nacre, indicating that nacre is composed of continuous parallel lamellae; bar, 2 lm. (B) Each nacre lamellae is composed of polygonal aragonitic tablets, which is sealed each other by intertabular matrix (arrow indicating); bar, 20 lm.
When injected into the extrapallial fluid between shell and mantle tissue, where nacre calcification occurs, the antibodies against a special matrix protein could interrupt the action of this protein in nacre calcification, resulting in aberrant growth of innermost lamella. The functions of this protein in nacre calcification will be indicated from the aberrant growth pattern. Nacrein is a major SM protein in the nacre of the pearl oyster Pinctada fucata, and has carbonic anhydrase (CA) activity (Miyamoto et al., 1996). CA activity is essential for rapid shell development (Wilbur and Jodrey, 1955; Freeman, 1960). Moreover, nacrein-like proteins are conserved in bivalves and gastropods, and thus are thought to play an important role in nacre mineralization of the two species (Miyamoto et al., 2005). In this study, we conduct immunolocalization and antibody inhibition assays to examine its function in nacre mineralization. We also characterize an ISM protein by the antibodies raised against the recombinant protein of P. fucata bone morphogenetic protein 2 (Pf-BMP-2). A better understanding of the structural and functional roles of SM and ISM proteins in nacre biomineralization will provide essential knowledge to mimic nacre. 2. Materials and methods 2.1. Animals The pearl oyster Pinctada fucata with shells of about 4.5–6.0 cm in diameter and 32–40 g in wet weight were obtained from Beihai, China and maintained in aerated artificial seawater at 3% salinity in an aquarium.
(NCA) (GenBank Accession No: BAA11940) were developed in New Zealand rabbits and BALB/c mice, respectively (Gong et al., 2008). The DNA encoding TGF-b domain (amino acids 346–447) of Pf-BMP-2 (GenBank Accession No. AB176952) was cloned into pET-28b expression vector (Novagen) and expressed in bacterial strain of Escherichia coli BL21 (DE3). The recombinant protein was highly purified using a Hitrap chelating HP affinity column (Amersham Biosciences). Polyclonal antibodies against the recombinant BMP-2 (anti-P43) or total SM proteins (anti-SM) were raised in New Zealand rabbits using a protocol as described by Mayer and Walker (1987). The antisera were then purified by 33% saturation ammonium sulfate precipitation and a protein A agarose column (Calbiochem). The purified antibodies were used for immunoblot analysis, immunolabeling and antibody inhibition assay. 2.4. Immunoblot analysis The proteins were electrophoretically transferred to nitrocellulose membranes using a Hoefer semidry blotter (Pharmacia Biotech). The nitrocellulose membranes were blocked with 5% skimmed milk and incubated with 5 lg/ml primary antibody for 2 h. After washing and incubated with an appropriate second antibody conjugated alkaline phosphatase (Santa Cruz), detection was carried out using BCIP/NBT solution (Roche). 2.5. Shell decalcification
The shell nacre from Pinctada fucata was ground and dissolved in 0.5 M EDTA (pH 8.0) for 4 days at 4 °C. After centrifugation, the supernatant containing SM proteins was collected for immunoblot analysis. The pellet containing ISM proteins was resuspended in 20 mM Tris–HCl (pH 7.0) containing 1% SDS, 10 mM DTT and boiled for 20 min. After centrifugation, the supernatant was precipitated in 85% ethanol at 4 °C. The precipitated proteins were washed twice with 85% ethanol, and redissolved in 20 mM Tris– HCl (pH 7.0) containing 6 M urea. The resulting solution was absorbed in a heparin agarose affinity column (Amersham Biosciences), washed thoroughly, and eluted with 20 mM Tris–HCl (pH 7.0) containing 0.1 M or 0.2 M NaCl.
The complete decalcification of nacre lamellae was performed as described (Nudelman et al., 2006). Briefly, the nacreous layers were mechanically isolated from the shell of P. fucata and decalcified at room temperature for 2 days in a solution containing 1 M EDTA (pH 8.0), 4% formaldehyde, and 0.5% cetylpyridinium chloride. The decalcified fragments were thoroughly washed with Milli-Q water. The lamellar sheets were peeled off from the fragments, mounted on glass slides, air dried and ready for immunolabeling. Partial decalcification of nacre lamellae was performed as follows. The fresh nacre was mechanically fractured into small fragments and cleaned in diluted sodium hypochlorite solution for 2 min (0.2 wt% active chlorine; Marin et al., 2007). The fragments were then partially etched by 10 mM acetic acid (pH 3.5) for 1– 3 min and neutralized with 10 mM sodium bicarbonate (Cho et al., 1996). The partially etched nacre lamellae were thoroughly washed with Milli-Q water and were ready for immunolabeling.
2.3. Preparation of antibodies
2.6. Immunolabeling of nacre lamellae
Rabbit polyclonal antibodies (P-anti-NCA) and a mouse monoclonal antibody (M-anti-NCA) against CA domain of nacrein
Completely decalcified lamellar sheets, as well as partially etched nacre lamellae, were blocked in 10% goat serum for 2 h,
2.2. Extraction and isolation of shell matrix proteins
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and incubated with a primary antibody at concentration of 5 lg/ ml or preimmune serum for 2 h. The samples were then incubated with an appropriate secondary antibody. Individual primary antibodies used were rabbit polyclonal antibodies that recognized SM proteins, nacrein or P43 (anti-SM, P-anti-NCA or anti-P43) or a mouse monoclonal antibody that recognized nacrein (M-anti-NCA). The secondary antibodies were goat anti-rabbit/mouse coupled to rhodamine (Santa Cruz) and goat antirabbit coupled to 12 nm gold particles (Jackson). In each step of immunolabeling, the samples were washed three times with PBS containing 0.05% Tween 20 for 5 min. At last, samples were washed in water. The rhodamine labeled samples were examined by a fluorescence microscope (Leica DIMR). The immunogold labeled samples were dried overnight, carbon coated and examined using a scanning electron microscopy (SEM) (Hitachi S-5500 UHR FE-SEM). 2.7. Antibody inhibition assay The purified antibodies were injected into the center of the extrapallial space between the mantle and the shell through the mantle with a microsyringe at the dosage of 0.5 lg (low dosage) and 1 lg (high dosage) per gram of wet weight per day. Each group is consist of four specimens (Ma et al., 2007). The oysters were sacrificed 3 days after antibody injection. The shells were separated, washed with Milli-Q water, and immersed in 5% NaOH for 8 h to remove organic components attached to the surfaces. The shells were then thoroughly washed with Milli-Q water, air dried, gold coated, and observed under a SEM (FEI Sirion2000). 3. Results 3.1. Immunoblot analysis of EDTA–soluble matrix (SM) proteins
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rein (M-anti-NCA) immunoreacted with a single band at about 55 kDa (Fig. 2B, lanes 1 and 2), which was consistent with the estimated molecular mass of nacrein on SDS–PAGE (Miyamoto et al., 1996; Takakura et al., 2008). These indicated that our antibodies specifically recognize nacrein. The polyclonal antibodies against total SM proteins (anti-SM) detected multiple bands, including a major band at around 55 kDa and several below 55 kDa in immunoblot analysis of SM proteins (Fig. 2B, lane 3). These results indicated that anti-SM had extensive immuno-reactivity with the proteins in SMs. 3.2. Immunoblot analysis of EDTA–insoluble matrix (ISM) proteins and characterization of a matrix protein P43 ISMs were extracted with a solution containing 1% SDS and 2 mM DTT by heating and redissolved in 6 M urea. The prepared proteins (Fig. 3A, lane 1) were loaded onto a heparin affinity column and eluted with 100 mM or 200 mM NaCl. On SDS–PAGE gel stained with Coomassie brilliant blue R250, a 43 kDa band was dominant in 100 mM NaCl-eluate (Fig. 3A, lane 2), and another 43 kDa band was prevailed in 200 mM NaCl-eluate (Fig. 3A, lane 3). Immunoblot analysis using polyclonal antibodies against TGF-b domain of Pf-BMP-2 (Fig. 3A, lane 4; Fig. 3B, lane 1) revealed a single band with molecular weight of 43 kDa (Fig. 3B, lanes 2 and 3) in ISM protein samples and 100 mM NaCl-eluate, while this band was not detected in 200 mM NaCl-eluate (Fig. 3B, lane 4). Preimmune serum did not recognize this 43 kDa band (Fig. 3B, lane 5). Furthermore, a weak band at 43 kDa (Fig. 3B, lane 6) was also observed in SM protein samples (Fig. 2A). Taken together, these results indicated that our antibodies specifically recognize a protein with molecular weight of 43 kDa. This protein is predominantly located in ISM, and a trace amount of it exists in SM. Based on its apparent molecular mass, we named it P43.
SM proteins were extracted from nacre powder with 0.5 M EDTA (Fig. 2A). Immunoblot analysis revealed that both polyclonal antibodies (P-anti-NCA) and a monoclonal antibody against nac-
3.3. Immunolabeling of matrix proteins in completely decalcified nacre lamellae
Fig. 2. SDS–PAGE (A) and immunoblot (B) analysis of EDTA–soluble matrix (SM) proteins extracted from nacre of P. fucata. (A) Lane 1: SDS–PAGE analyzing SM proteins stained by Coomassie brilliant blue R-250; M, molecular weight standards, kDa. (B) Lanes 1–3: immunoblot analyzing SM proteins with polyclonal antibodies and a monoclonal antibody against nacrein, polyclonal antibodies against SM proteins, respectively.
Nacre lamella is composed of polygonal aragonitic tablets with widths ranging between 3 and 7 lm (Fig. 1B). The space between each aragonitic tablet is sealed by intertabular matrix (arrows, Fig. 1B). The central region of crystal imprints was supposed to be the site of aragonite nucleation (Rousseau et al., 2005b; Nudelman et al., 2006). After complete decalcification of lamellae, only organic interlamellar sheets remained, and individual crystal imprints on the sheets were no longer detected by a microscope (Fig. 4A). As high molecular weight of antibodies complex for immunolabeling makes them hardly diffuse through the sheets, immunolabeling of matrix proteins in decalcified nacre lamellae mostly reflects their distributions in the surface layer of interlamellar sheets (Nudelman et al., 2006). Immunolabeling experiment showed that SM proteins existed in polygonal crystal imprints in the organic matrix sheet (arrowhead) as well as the central region of the imprints (arrow, Fig. 4C), suggesting that SM proteins constitute the intertabular matrix and the nucleation sites (Nudelman et al., 2006). Immunolabeling using P-anti-NCA and M-anti-NCA both discerned polygonal crystal imprints (arrowheads), but indiscernible staining was observed in the central area (Fig. 4D and E), indicating that nacrein may be localized to intertabular matrix. Different from anti-NCA labeling, anti-P43 labeling did not outline polygonal crystal imprints, but discerned fluorescent spots widely distributing in the matrix sheet (Fig. 4E). This suggested that P43 may be in the aragonitic tablets or localized to the interlamellar sheet. In the negative control of immunolabeling, no staining was shown in the matrix sheet (Fig. 4B).
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Fig. 3. SDS–PAGE (A) and immunoblot (B) analysis of EDTA–insoluble matrix (ISM) proteins extracted from nacre of P. fucata. (A): Lane 1, SDS–extracted ISM proteins redissolved in 6 M urea; lane 2, 100 mM NaCl-eluate (E1) from a heparin affinity column; lane 3, 200 mM NaCl-eluate (E2) from the column; lane 4, the purified recombinant protein encoding the TGF-b domain of Pf-BMP-2, indicated by arrow; M, molecular weight standards, kDa. (B): Lanes 1–4 and 6, immunoblot analyzing the recombinant protein of Pf-BMP-2 (about 14 kDa), ISM proteins, E1, E2, and SM proteins (Fig. 2A, lane 1) with anti-P43 as primary antibody, respectively; lane 5, the negative control, analyzing ISM proteins with preimmune serum; arrows indicating the visualized bands.
Fig. 4. (A) Light micrograph of completely decalcified interlamellar sheets, in which only organic matrix remained; the lines in the picture are due to folds in the interlamellar sheet. (B–F) fluorescence micrographs of decalcified interlamellar sheets labeled by preimmune serum, anti-SM, P-anti-NCA, M-anti-NCA and anti-P43, respectively. Arrowheads indicate the stained intertabular matrix. Arrow in (C) indicates the stained area in the center of the crystal imprints. Bar, 20 lm.
3.4. Immunogold labeling of matrix proteins in partially etched nacre lamellae Immunogold labeling partially etched nacre lamellae will reveal the distributions of matrix proteins in aragonitic tablets, which is observed by a SEM in back-scattered electron mode (SEM-BSE). In this model, elements with higher atomic number scatter more electrons thus appearing lighter. Therefore, immunogold particles are displayed as the brightest tiny spots; the mineral phase is lighter than the organic phase. Fig. 5C and D (arrow indicating) revealed that nacrein actually distributes in intertabular matrix. Figs. 5C and D and 6C and D (arrowhead indicating) showed that this protein also widely exists in the aragonitic tablets and seems to attach to the mineral phase. As an ISM, P43 is rarely localized to the intertabular matrix (Fig. 5E and F), but exists in aragonitic tablets (arrowhead indicating, Figs. 5E and F and 6E and F). The negative control (Figs. 5A and B and 6A and B) with preimmune serum suggested that the positive staining is reliable. Fig. 7 showed the distributions of nacrein and P43 in the crosssection of nacre lamellae (section parallel to the c-axis). The immunogold labeling confirmed that nacrein widely distributes in nacre lamellae and is embedded in the mineral phase as calcium carbon-
ate crystals growing (Fig. 7C and D). P43 distributes within the lamellae (Fig. 7E and F, black arrows), and also is localized to interlamellar matrix at interface with the mineral phase (Fig. 7E and F, white arrows). As expected, the negative controls displayed a low background, which make the labeling reliable (Fig. 7A and B). 3.5. In vivo investigation of the actions of nacrein and P43 in the growth of aragonitic tablets There is a space between nacre and mantle tissue, filled with body fluid. Matrix proteins secreted by mantle tissue are released into this space and act in the growth of nacre lamellae. The purified antibodies against P43 and nacrein were injected into this space to disrupt the functions of these proteins in nacre growth. Each antibody-injected group contained four oysters. As a control, preimmune rabbit serum was injected at same dosage. Three days later, the oysters were sacrificed. The inner surfaces of the nacre were examined by a SEM. 3.5.1. Treatment of anti-P43 In low dosage anti-P43 injected-group, the aragonite nanograins failed to pack into a compact aggregation as the untreated ones (Fig. 8C), and became disordered (Fig.8C and D). As the injection dos-
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Fig. 5. Immunogold labeling matrix proteins in the intertabular matrix. (A, C, and E) Labeling the aragonitic tablets with preimmune serum, P-anti-NCA, and anti-P43, respectively. (B, D, and E) SEM-BSE observing immunogold staining in the boxes of (A, C, and E) respectively; arrows indicating immunogold particles localized to the intertabular matrix; arrowheads indicating the gold particles on the aragonitic tablets. Bars, (A–F) 2 lm, 400 nM, 3 lm, 500 nm, 3 lm, 400 nm, respectively.
Fig. 6. Immunogold labeling matrix proteins within partially etched aragonitic tablets. (A, C, and E) labeling the aragonitic tablets with preimmune serum, P-anti-NCA, and anti-P43, respectively. (B, D, and E) SEM-BSE imaging immunogold staining in the boxes of (A, C, and E) respectively; arrowheads indicating immunogold particles distributing in the aragonitic tablets. Bars, (A–F) 3 lm, 500 nM, 3 lm, 500 nm, 4 lm, 400 nm, respectively.
age doubled, the growth of aragonitic tablets was seriously disrupted. Calcium carbonate crystals with organic substance failed to assemble into aragonitic tablets within 4 4 mm2 area around the injection site (Fig. 8E and F). In contrast, aragonitic tablets of preimmune serum treated group kept the normal shape and framework (Figs. 8A and B and 9D–F). These observations indicated that antiP43 treatment disrupted the organo-mineral assemblage of aragonitic tablets, suggesting that P43 should be a framework matrix. 3.5.2. Treatment of anti-NCA in high dosage Although the aragonitic tablets appeared coarser by preimmune serum treatment than untreated ones (Fig. 9A–C), the thickness had little change (Fig. 9D–F). In contrast, P-anti-NCA treatment resulted in thickening of aragonitic tablets (Fig. 9G–I). While the normal tablets displayed uniformed surface (Fig. 9B and C), aberrant tablets did not. The surface of aberrant tablets was characterized as an accumulation of inconsistent nanograins (Fig. 9H and I). Moreover, calcium carbonate crystals precipitated randomly out-
side the aberrant aragonitic tablets. In about 4 4 mm2 area around injection sites, aberrant growth of aragonite crystal occurred in all 4 experimental oysters. Similarly, aberrant aragonitic tablets, which thickened, were also observed in the inner lamellae of M-anti-NCA injected group (Fig. 9J–L). The aberrant tablets exhibited inconsistent nanograins. Less calcium carbonate crystals were deposited around aragonitic tablets in M-anti-NCA treated group (Fig. 9K) than P-anti-NCA treated group (Fig. 9H). It is noticeable that aragonitic tablets in anti-NCA injected groups had normal polygonal shape and framework. These observations suggested that nacrein mostly functions in the regulation of the growth of calcium carbonate crystals.
4. Discussion In this study, we three-dimensionally map the distributions of SM proteins and an ISM protein in nacre lamellae, and conduct
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Fig. 7. Immunogold labeling the cross-section of partially etched nacre lamellae (section parallel to the c-axis). (A, C, and E) The lamellae labeled by preimmune serum, Panti-NCA, and anti-P43, respectively; (B, D, and F) SEM-BSE imaging the immunogold staining in the lamellae of (A, C, and E) respectively. Bars, (A and B) 500 nm; (C and D) 400 nm; (E and F) 500 nm.
Fig. 8. (A) SEM image of innermost lamella in low dosage preimmune serum treated group; (B) enlargement of the box in (A); (C) SEM image of innermost lamella in low dosage anti-P43 treated group; (D) enlargement of the box in (C) showing defective aragonitic tablets; (E) SEM image of innermost lamella in high dosage anti-P43 treated group; (F) enlargement of the box in (E) showing calcium carbonate crystals adhering with organic substance. Bars, (A, C, and E) 20 lm; (B, D, and F) 2 lm.
antibody inhibition assay to study their functions in nacre calcification. The understanding of the localization of matrix proteins is central to understanding their functions in the growth of nacre lamellae. It is known that SM proteins precisely regulate nacre mineralization, therefore control crystal morphology, crystal phase switching and orientation (Mann et al., 1993; Belcher et al., 1996; Levi et al., 1998). Herein, our immunolabeling shows that SM proteins are localized to the central region and the periphery of polygonal crystal imprints in interlamellar sheets (Fig. 4C), suggesting that SM proteins are localized to the nucleation sites of aragonitic tablets and the intertabular matrix (Crenshaw and Ristedt, 1976; Rousseau et al., 2005b; Nudelman et al., 2006). As a major component in SM proteins, nacrein is also demonstrated its distribution in the intertabular matrix by anti-NCA labeling (Figs. 4D and E and 5C and D). Since intertabular matrix associated proteins are
thought to function in the inhibition or cessation of crystal growth (Marin et al., 2000; Nudelman et al., 2006), the distribution of nacrein in intertabular matrix might suggest its function in the inhibition or cessation of crystal growth. By calcium carbonate precipitation assay, Miyamoto et al. (2005) also suggested that nacrein might act as a negative regulator in shell calcification. To learn the actual role of nacrein in the growth of nacre lamellae, we conducted antibody inhibition assays using rabbit polyclonal antibodies and a mouse monoclonal antibody against the CA domain of nacrein. A CA-dependent role of nacrein in the regulation of calcium carbonate crystal growth is revealed. In both antibodies treatment groups, the most obvious change is the thickening of aragonitic tablets resulting from disordered nanograins growth, which is distinct from the normal ones assembled by coherent nanograins with consistent crystal orientation (Rousseau et al., 2005a). Considering its wide distribution in aragonitic
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Fig. 9. (A–C) SEM images of untreated aragonitic tablets;(D–F) SEM images of innermost lamella in high dosage preimmune serum treated group; (G–I) SEM images of innermost lamella in high dosage P-anti-NCA treated group; (J–L) SEM images of innermost lamella in high dosage M-anti-NCA treated group. (B, E, H, and K) enlargement of the boxes in (A, D, G, and J) respectively. (C, F, I, and L) enlargement of the boxes in (B, E, H, and K) respectively. (C) The smooth surface of normal aragonitic tablets; (F) High dosage preimmune serum treatment resulting aragonitic tablets with coarser surface; (I and L) P-anti-NCA and M-anti-NCA treatments resulting thickening aragonitic tablets with inconsistent nanograins. Bars, (A, D, G, and J) 50 lm; (B, E, H, and K) 5 lm; (C, F, I, and L) 1 lm.
tablets (Fig. 7C and D), we believe that nacrein with CA activity likely functions as a negative regulator in the growth of nanogains in aragonitic tablets, ensuring their consistent crystal orientation and growth rate. Since CA catalyzes the reversible hydration of CO2 into HCO3 (Tripp et al., 2001), nacrein might exert its regulatory function via controlling carbonate/bicarbonate equilibrium for calcium carbonate precipitation or adjusting pH in the microenvironment of calcification. Furthermore, nacrein widely distributes over the surface of aragonitic tablets (Fig. 6C and D). This distribution allows nacrein to act as an inhibiting surface, constraining crystals growth in the c-axis direction. Therefore, as nacrein is suppressed in vivo, crystal overgrows in the c-axis direction (Fig. 9H and K). Moreover, we notice that more undesirable crystals form around aragonitic tablets after P-anti-NCA treatment (Fig. 9H) than after M-anti-NCA treatment (Fig. 9K). This could be due to the fact that more epitopes in CA domain are recognized and blocked by polyclonal antibodies than by a monoclonal antibody. These observations indicate that the negative regulation would also prevent calcium carbonate crystallizing in undesirable sites. As an ISM protein, the distribution of P43 is quite different from SM proteins. In completely decalcified lamellae, anti-P43 label is
not detectable in the periphery of crystal imprints as does antiSM, but is discerned as many fluorescent spots on the organic sheet (Fig. 4F). This suggests that P43 might be absorbed in the interlamellar sheet. In the cross-section of nacre lamellae, the immunogold labeling reveals that P43 is actually localized to interlamellar matrix sheet (Fig. 7E and F), and also embedded within aragonitic tablets (Figs. 6E, F and 7E and F). Antibody inhibition assay confirms a role of P43 in the construction of organic framework of nacre lamellae. When P43 was suppressed in vivo, calcium carbonate crystals adhering to fiber-like organic substances, but not normal aragonitic tablets, grew toward the innermost lamella (Fig. 8E and F). This observation shows that P43 is necessary for the buildup of aragonitic tablets. According to its distribution in aragonitic tablets, we suppose that this protein should participate in the buildup of the continuous organic matrix framework within aragonitic tablets as Rousseau et al. (2005a) observed, supporting coherent aggregation of nanograins. Its ability to bind heparin makes it possible to connect with other matrix within aragonitic tablets and the interlamellar matrix, for instance chitin, silk fibroin and some proteoglycans (Ruppert et al., 1996; Levi-Kalisman et al., 2001; Addadi et al., 2006).
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Since the discovery that nacre powder can induce bone formation in vivo (Westbroek and Marin, 1998), BMP homologs are hypothesized to exist in the matrix of the nacreous shell layer. P43, which is a matrix protein, can be specifically recognized by the antibodies against the recombinant protein encoding the TGF-b domain of Pf-BMP-2. This suggests that there are some homological epitopes expressed in P43 and TGF-b domain of PfBMP-2. Similar to mammalian BMP-2 (Ruppert et al., 1996), P43 exhibits an affinity for heparin in a heparin affinity chromatography. Immunoblot analysis and affinity chromatography reveal some features of P43. Future work will involve purification of P43. When fully characterized, P43 could be proved important not only in biomineralogy but also in medicine and evolutionary biology. Our results show that SM and ISM proteins differentially distribute in nacre lamellae and function differently in nacre mineralization. Some regulating activities of matrix proteins in the growth of aragonitic tablets would be indicated by antibody inhibition assays. The analysis of aberrant growth pattern of aragonitic tablets along with immunolocalization would further reveal the regulatory mechanisms of matrix proteins in nacre mineralization. Acknowledgments This work was financially supported by the National High Technology Research and Development Program of China (2003AA603430, 2006AA09Z413) and the Natural Science Foundation of China (30221003, 30530600). We are grateful to Dr. Yan Ma, Dana-Farber Cancer Institute, Boston, for critical discussion. References Addadi, L., Joester, D., Nudelman, F., Weiner, S., 2006. Mollusk shell formation: a source of new concepts for understanding biomineralization processes. Chem. Eur. J. 12, 980–987. Belcher, A.M., Wu, X.H., Christensen, R.J., Hansma, P.K., Stucky, G.D., Morse, D.E., 1996. Control of crystal phase switching and orientation by soluble molluscshell proteins. Nature 381, 56–58. Cho, J.W., Partin, J.S., Lennnarz, W.J., 1996. A technique for detecting matrix proteins in the crystalline spicules of the sea urchin embryo. Proc. Natl. Acad. Sci. USA 93, 1282–1286. Crenshaw, M.A., 1972. The soluble matrix from Mercenaria mercenaria shell. Biomineralization 6, 6–11. Crenshaw, M.A., Ristedt, H., 1976. The histochemical localization of reactive groups in septal nacre from Nautilus pompilius. In: Watabe, N., Wilbur, K.M. (Eds.), The Mechanisms of Mineralization in the Invertebrates and Plants. University of South Carolina Press, Colombia, pp. 355–367. Freeman, J.A., 1960. Influence of carbonic anhydrase inhibitors on shell growth of freshwater snail Physa heterostropha. Biol. Bull 118, 412–418. Gong, N., Li, Q., Huang, J., Fang, Z., Xie, L., Zhang, R., 2008. Culture of outer epithelial cells from the mantle tissue to study matrix protein secretion for biomineralization. Cell Tissue Res. doi:10.1007/s00441-008-0609-5. Lamghari, M., Almeida, M.J., Berland, S., Huet, H., Laurent, A., Milet, C., Lopez, E., 1999. Stimulation of bone marrow cells and bone formation by nacre: in vivo and in vitro studies. Bone 25 (Suppl. 2), 91S–99S. Levi, Y., Albeck, S., Brack, A., Weiner, S., Addadi, L., 1998. Control over aragonite crystal nucleation and growth: an in vitro study of biomineralization. Chem. Eur. J. 4, 389–396.
Levi-Kalisman, Y., Falini, G., Addadi, L., Weiner, S., 2001. Structure of the nacreous organic matrix of a bivalve mollusk shell examined in the hydrated state using cryo-TEM. J. Struct. Biol. 135, 8–17. Ma, Z., Huang, J., Sun, J., Wang, G., Li, C., Xie, L., Zhang, R., 2007. A novel extrapallial fluid protein controls the morphology of nacre lamellae in the pearl oyster, Pinctada fucata. J. Biol. Chem. 282, 23253–23263. Mann, S., Archibald, D.D., Didymus, J.M., Douglas, T., Heywood, B.R., Meldrum, F.C., Reeves, N.J., 1993. Crystallization at inorganic–organic interfaces: biominerals and biomimetic synthesis. Science 261, 1286–1292. Marin, F., de Corstjens, P., de Gaulejac, B., Vrind-De, Jong E., Westbroek, P., 2000. Mucins and molluscan calcification. Molecular characterization of mucoperlin, a novel mucin-like protein from the nacreous shell layer of the fan mussel Pinna nobilis (Bivalvia, pteriomorphia). J. Biol. Chem. 275, 20667–20675. Marin, F., Pokroy, B., Luqueta, G., Layrollec, P., Groot, K.D., 2007. Protein mapping of calcium carbonate biominerals by immunogold. Biomaterials 28, 2368–2377. Mayer, R.J., Walker, J.H., 1987. Immunochemical Methods in Cell and Molecular Biology. Academic Press, London. Miyamoto, H., Miyashita, T., Okushima, M., Nakano, S., Morita, T., Matsushiro, A., 1996. A carbonic anhydrase from the nacreous layer in oyster pearls. Proc. Natl. Acad. Sci. USA 93, 9657–9660. Miyamoto, H., Miyoshi, F., Kohno, J., 2005. The carbonic anhydrase domain protein nacrein is expressed in the epithelial cells of the mantle and acts as a negative regulator in calcification in the mollusk Pinctada fucata. Zool. Sci. 22, 311–315. Mouriès, L.P., Almeida, M.J., Milet, C., Berland, S., Lopez, E., 2002. Bioactivity of nacre water-soluble organic matrix from the bivalve mollusk Pinctada maxima in three mammalian cell types: fibroblasts, bone marrow stromal cells and osteoblasts. Comp. Biochem. Physiol. B Biochem. Mol. Biol. 132, 217–229. Nudelman, F., Gotliv, B.A., Addadi, L., Weiner, S., 2006. Mollusk shell formation: mapping the distribution of organic matrix components underlying a single aragonitic tablet in nacre. J. Struct. Biol. 153, 176–187. Puverel, S., Tambutté, E., Zoccola, D., Domart-Coulon, I., Bouchot, A., Lotto, S., Allemand, D., Tambutté, S., 2005. Antibodies against the organic matrix in scleractinians: a new tool to study coral biomineralization. Coral Reefs 24, 149– 156. Rousseau, M., Lopez, E., Stempflé, P., Brendlé, M., Franke, L., Guette, A., Naslain, R., Bourrat, X., 2005a. Multiscale structure of sheet nacre. Biomaterials 26, 6254– 6262. Rousseau, M., Lopez, E., Couté, A., Mascarel, G., Smith, D.C., Naslain, R., Bourrat, X., 2005b. Sheet nacre growth mechanism: a Voronoi model. J. Struct. Biol. 149, 149–157. Ruppert, R., Hoffmann, E., Sebald, W., 1996. Human bone morphogenetic protein 2 contains a heparin-binding site which modifies its biological activity. Eur. J. Biochem. 237, 295–302. Silve, C., Lopez, E., Vidal, B., Smith, D.C., Camprasse, S., Camprasse, G., Couly, G., 1992. Nacre initiates biomineralization by human osteoblasts maintained in vitro. Calcif. Tissue Int. 1, 363–369. Stolarski, J., Mazur, M., 2005. Nanostructure of biogenic versus abiogenic calcium carbonate crystals. Acta Palaeontol. Pol. 50, 847–865. Sudo, S., Fujikawa, T., Nagakura, T., Ohkubo, T., Sakaguchi, K., Tanaka, M., Nakashima, K., Takahashi, T., 1997. Structures of mollusc shell framework proteins. Nature 387, 563–564. Takakura, D., Norizuki, M., Ishikawa, F., Samata, T., 2008. Isolation and characterization of the N-linked oligosaccharides in nacrein from Pinctada fucata. Mar. Biotechnol. (NY) 10, 290–296. Tripp, B.C., Smith, K., Ferry, J.G., 2001. Carbonic anhydrase: new insights for an ancient enzyme. J. Biol. Chem. 276, 48615–48618. Wada, K., 1968. Mechanism of growth of nacre in bivalvia. Bull. Natl. Pearl. Res. Lab. 13, 1561–1596. Weiner, S., Addadi, L., 1997. Design strategies in mineralized biological materials. J. Mater. Chem. 7, 689–702. Weiner, S., Gotliv, B., Levi-Kalisman, Y., Raz, S., Weiss, I. M., Addadi, L., 2003. Biomineralization (BIOM2001): formation, diversity, evolution and application. In: Kobayashi, I., Ozawa, H. (Eds.), Proceedings of the 8th International Symposium on Biomineralization. Tokai University Press, Kanagawa, pp. 8-13. Westbroek, P., Marin, F., 1998. A marriage of bone and nacre. Nature 392, 861–862. Wilbur, K.M., Jodrey, L., 1955. Studies of shell formation. V. The inhibition of shell formation by carbonic anhydrase inhibitors.. Biol. Bull. (Woods Hole) 108, 82– 112. Zhang, C., Zhang, R., 2006. Matrix proteins in the outer shells of molluscs. Mar. Biotechnol. (NY) 8, 572–586.
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Immunolocalization of matrix proteins in nacre lamellae and their in vivo effects on aragonitic tablet growth Ningping Gong a, Junlong Shangguan a, Xiaojun Liu a, Zhenguang Yan a, Zhuojun Ma a, Liping Xie a,b,*, Rongqing Zhang a,b,* a b
Institute of Marine Biotechnology, Department of Biological Sciences and Biotechnology, Tsinghua University, Beijing 100084, China Protein Science Laboratory of the Ministry of Education, Tsinghua University, Beijing 100084, China
a r t i c l e
i n f o
Article history: Received 9 January 2008 Received in revised form 23 May 2008 Accepted 24 May 2008 Available online 3 June 2008 Keywords: Mollusk shell nacre Matrix protein BMP-2 Nacrein Antibodies
a b s t r a c t How matrix proteins precisely control the growth of nacre lamellae is an open question in biomineralization research. Using the antibodies against matrix proteins for immunolabeling and in vivo experiments, we investigate the structural and functional roles of EDTA–soluble matrix (SM) and EDTA– insoluble matrix (ISM) proteins in nacre biomineralization of the pearl oyster Pinctada fucata. Immunolabeling reveals that a SM protein, nacrein, distributes within aragonitic tablets and intertabular matrix. An ISM protein, which we named P43, has been specifically recognized by polyclonal antibodies raised against the recombinant protein of P. fucata bone morphogenetic protein 2 in immunoblot analysis. Immunolabeling indicates that P43 is localized to interlamellar sheet, and also embedded within aragonitic tablets. Although nacrein and P43 both distribute within aragonitic tablets, they function differently in aragonitic tablet growth. When nacrein is suppressed by the antibodies against it in vivo, crystal overgrowth occurs, indicating that this SM protein is a negative regulator in aragonitic tablet growth. When P43 is suppressed in vivo, the organo-mineral assemblage is disrupted, suggesting that P43 is a framework matrix. Taken together, SM and ISM proteins are indispensable factors for the growth of nacre lamellae, controlling crystal growth and constructing the framework of aragonitic tablets. Ó 2008 Elsevier Inc. All rights reserved.
1. Introduction Nacre (mother-of-pearl) is a biogenetic material widely distributing in mollusks, and usually forms in the inner layer of shells. The structure of nacre from bivalves is characterized as an arrangement of continuous parallel lamellae (Fig. 1A), separated by sheets of interlamellar matrix (Wada, 1968; Addadi et al., 2006). Each lamella is composed of polygonal aragonitic tablets (Fig. 1B), which is sealed to each other by intertabular matrix. An aragonitic tablet seems to be a single crystal, but actually is a coherent aggregation of crystalline nanograins (about 45 nm mean size) with the same crystallographic orientation, connected by a continuous organic framework (Rousseau et al., 2005a; Stolarski and Mazur, 2005). Nacre is an organo-mineral assemblage with dominant calcium carbonate and a minor organic matrix complex, resulting in superior mechanical properties and excellent osteoinductive activity (Silve et al., 1992; Lamghari et al., 1999; Weiner et al., 2003). The organic matrix complex includes proteins and polysaccharides, which is thought to direct the growth of calcium carbonate crystal * Corresponding authors. Fax: +86 10 62772899. E-mail addresses: [email protected] (L. Xie), [email protected] (R. Zhang). 1047-8477/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.jsb.2008.05.009
and be responsible for the extraordinary properties of nacre (Weiner and Addadi, 1997; Mouriès et al., 2002; Zhang and Zhang, 2006). Matrix proteins in nacre are usually classified into two groups depending on their solubility in decalcifying solution (Crenshaw and Ristedt, 1976). EDTA-based solution is often used for decalcification. EDTA–soluble matrix (SM) proteins have been proposed to localize within calcium carbonate crystals as intracrystalline matrix (Crenshaw, 1972) and control crystal morphology, crystal phase switching and orientation (Mann et al., 1993; Belcher et al., 1996; Zhang and Zhang, 2006). In contrast, EDTA–insoluble matrix proteins (ISMs) are hypothesized to localize around calcium carbonate crystallites and thus are supposed as intercrystalline matrices and framework macromolecules (Crenshaw, 1972; Sudo et al., 1997). Matrix proteins play vital roles in the formation of nacre. But how matrix proteins interact with the mineral in the self-organization of nacre has not been fully understood yet. Immunolocalization has been widely used to study the functions of matrix proteins in the growth of biomaterials, such as sea urchin, coral skeletons and shell (Crenshaw and Ristedt, 1976; Cho et al., 1996; Puverel et al., 2005; Nudelman et al., 2006; Marin et al., 2007). Antibody inhibition assay is proved to be a reliable method for in vivo investigation of the functions of matrix proteins in the growth of nacre lamellae (Ma et al., 2007).
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Fig. 1. Nacre structure of P. fucata. (A) SEM view shows the cross-section of nacre, indicating that nacre is composed of continuous parallel lamellae; bar, 2 lm. (B) Each nacre lamellae is composed of polygonal aragonitic tablets, which is sealed each other by intertabular matrix (arrow indicating); bar, 20 lm.
When injected into the extrapallial fluid between shell and mantle tissue, where nacre calcification occurs, the antibodies against a special matrix protein could interrupt the action of this protein in nacre calcification, resulting in aberrant growth of innermost lamella. The functions of this protein in nacre calcification will be indicated from the aberrant growth pattern. Nacrein is a major SM protein in the nacre of the pearl oyster Pinctada fucata, and has carbonic anhydrase (CA) activity (Miyamoto et al., 1996). CA activity is essential for rapid shell development (Wilbur and Jodrey, 1955; Freeman, 1960). Moreover, nacrein-like proteins are conserved in bivalves and gastropods, and thus are thought to play an important role in nacre mineralization of the two species (Miyamoto et al., 2005). In this study, we conduct immunolocalization and antibody inhibition assays to examine its function in nacre mineralization. We also characterize an ISM protein by the antibodies raised against the recombinant protein of P. fucata bone morphogenetic protein 2 (Pf-BMP-2). A better understanding of the structural and functional roles of SM and ISM proteins in nacre biomineralization will provide essential knowledge to mimic nacre. 2. Materials and methods 2.1. Animals The pearl oyster Pinctada fucata with shells of about 4.5–6.0 cm in diameter and 32–40 g in wet weight were obtained from Beihai, China and maintained in aerated artificial seawater at 3% salinity in an aquarium.
(NCA) (GenBank Accession No: BAA11940) were developed in New Zealand rabbits and BALB/c mice, respectively (Gong et al., 2008). The DNA encoding TGF-b domain (amino acids 346–447) of Pf-BMP-2 (GenBank Accession No. AB176952) was cloned into pET-28b expression vector (Novagen) and expressed in bacterial strain of Escherichia coli BL21 (DE3). The recombinant protein was highly purified using a Hitrap chelating HP affinity column (Amersham Biosciences). Polyclonal antibodies against the recombinant BMP-2 (anti-P43) or total SM proteins (anti-SM) were raised in New Zealand rabbits using a protocol as described by Mayer and Walker (1987). The antisera were then purified by 33% saturation ammonium sulfate precipitation and a protein A agarose column (Calbiochem). The purified antibodies were used for immunoblot analysis, immunolabeling and antibody inhibition assay. 2.4. Immunoblot analysis The proteins were electrophoretically transferred to nitrocellulose membranes using a Hoefer semidry blotter (Pharmacia Biotech). The nitrocellulose membranes were blocked with 5% skimmed milk and incubated with 5 lg/ml primary antibody for 2 h. After washing and incubated with an appropriate second antibody conjugated alkaline phosphatase (Santa Cruz), detection was carried out using BCIP/NBT solution (Roche). 2.5. Shell decalcification
The shell nacre from Pinctada fucata was ground and dissolved in 0.5 M EDTA (pH 8.0) for 4 days at 4 °C. After centrifugation, the supernatant containing SM proteins was collected for immunoblot analysis. The pellet containing ISM proteins was resuspended in 20 mM Tris–HCl (pH 7.0) containing 1% SDS, 10 mM DTT and boiled for 20 min. After centrifugation, the supernatant was precipitated in 85% ethanol at 4 °C. The precipitated proteins were washed twice with 85% ethanol, and redissolved in 20 mM Tris– HCl (pH 7.0) containing 6 M urea. The resulting solution was absorbed in a heparin agarose affinity column (Amersham Biosciences), washed thoroughly, and eluted with 20 mM Tris–HCl (pH 7.0) containing 0.1 M or 0.2 M NaCl.
The complete decalcification of nacre lamellae was performed as described (Nudelman et al., 2006). Briefly, the nacreous layers were mechanically isolated from the shell of P. fucata and decalcified at room temperature for 2 days in a solution containing 1 M EDTA (pH 8.0), 4% formaldehyde, and 0.5% cetylpyridinium chloride. The decalcified fragments were thoroughly washed with Milli-Q water. The lamellar sheets were peeled off from the fragments, mounted on glass slides, air dried and ready for immunolabeling. Partial decalcification of nacre lamellae was performed as follows. The fresh nacre was mechanically fractured into small fragments and cleaned in diluted sodium hypochlorite solution for 2 min (0.2 wt% active chlorine; Marin et al., 2007). The fragments were then partially etched by 10 mM acetic acid (pH 3.5) for 1– 3 min and neutralized with 10 mM sodium bicarbonate (Cho et al., 1996). The partially etched nacre lamellae were thoroughly washed with Milli-Q water and were ready for immunolabeling.
2.3. Preparation of antibodies
2.6. Immunolabeling of nacre lamellae
Rabbit polyclonal antibodies (P-anti-NCA) and a mouse monoclonal antibody (M-anti-NCA) against CA domain of nacrein
Completely decalcified lamellar sheets, as well as partially etched nacre lamellae, were blocked in 10% goat serum for 2 h,
2.2. Extraction and isolation of shell matrix proteins
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and incubated with a primary antibody at concentration of 5 lg/ ml or preimmune serum for 2 h. The samples were then incubated with an appropriate secondary antibody. Individual primary antibodies used were rabbit polyclonal antibodies that recognized SM proteins, nacrein or P43 (anti-SM, P-anti-NCA or anti-P43) or a mouse monoclonal antibody that recognized nacrein (M-anti-NCA). The secondary antibodies were goat anti-rabbit/mouse coupled to rhodamine (Santa Cruz) and goat antirabbit coupled to 12 nm gold particles (Jackson). In each step of immunolabeling, the samples were washed three times with PBS containing 0.05% Tween 20 for 5 min. At last, samples were washed in water. The rhodamine labeled samples were examined by a fluorescence microscope (Leica DIMR). The immunogold labeled samples were dried overnight, carbon coated and examined using a scanning electron microscopy (SEM) (Hitachi S-5500 UHR FE-SEM). 2.7. Antibody inhibition assay The purified antibodies were injected into the center of the extrapallial space between the mantle and the shell through the mantle with a microsyringe at the dosage of 0.5 lg (low dosage) and 1 lg (high dosage) per gram of wet weight per day. Each group is consist of four specimens (Ma et al., 2007). The oysters were sacrificed 3 days after antibody injection. The shells were separated, washed with Milli-Q water, and immersed in 5% NaOH for 8 h to remove organic components attached to the surfaces. The shells were then thoroughly washed with Milli-Q water, air dried, gold coated, and observed under a SEM (FEI Sirion2000). 3. Results 3.1. Immunoblot analysis of EDTA–soluble matrix (SM) proteins
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rein (M-anti-NCA) immunoreacted with a single band at about 55 kDa (Fig. 2B, lanes 1 and 2), which was consistent with the estimated molecular mass of nacrein on SDS–PAGE (Miyamoto et al., 1996; Takakura et al., 2008). These indicated that our antibodies specifically recognize nacrein. The polyclonal antibodies against total SM proteins (anti-SM) detected multiple bands, including a major band at around 55 kDa and several below 55 kDa in immunoblot analysis of SM proteins (Fig. 2B, lane 3). These results indicated that anti-SM had extensive immuno-reactivity with the proteins in SMs. 3.2. Immunoblot analysis of EDTA–insoluble matrix (ISM) proteins and characterization of a matrix protein P43 ISMs were extracted with a solution containing 1% SDS and 2 mM DTT by heating and redissolved in 6 M urea. The prepared proteins (Fig. 3A, lane 1) were loaded onto a heparin affinity column and eluted with 100 mM or 200 mM NaCl. On SDS–PAGE gel stained with Coomassie brilliant blue R250, a 43 kDa band was dominant in 100 mM NaCl-eluate (Fig. 3A, lane 2), and another 43 kDa band was prevailed in 200 mM NaCl-eluate (Fig. 3A, lane 3). Immunoblot analysis using polyclonal antibodies against TGF-b domain of Pf-BMP-2 (Fig. 3A, lane 4; Fig. 3B, lane 1) revealed a single band with molecular weight of 43 kDa (Fig. 3B, lanes 2 and 3) in ISM protein samples and 100 mM NaCl-eluate, while this band was not detected in 200 mM NaCl-eluate (Fig. 3B, lane 4). Preimmune serum did not recognize this 43 kDa band (Fig. 3B, lane 5). Furthermore, a weak band at 43 kDa (Fig. 3B, lane 6) was also observed in SM protein samples (Fig. 2A). Taken together, these results indicated that our antibodies specifically recognize a protein with molecular weight of 43 kDa. This protein is predominantly located in ISM, and a trace amount of it exists in SM. Based on its apparent molecular mass, we named it P43.
SM proteins were extracted from nacre powder with 0.5 M EDTA (Fig. 2A). Immunoblot analysis revealed that both polyclonal antibodies (P-anti-NCA) and a monoclonal antibody against nac-
3.3. Immunolabeling of matrix proteins in completely decalcified nacre lamellae
Fig. 2. SDS–PAGE (A) and immunoblot (B) analysis of EDTA–soluble matrix (SM) proteins extracted from nacre of P. fucata. (A) Lane 1: SDS–PAGE analyzing SM proteins stained by Coomassie brilliant blue R-250; M, molecular weight standards, kDa. (B) Lanes 1–3: immunoblot analyzing SM proteins with polyclonal antibodies and a monoclonal antibody against nacrein, polyclonal antibodies against SM proteins, respectively.
Nacre lamella is composed of polygonal aragonitic tablets with widths ranging between 3 and 7 lm (Fig. 1B). The space between each aragonitic tablet is sealed by intertabular matrix (arrows, Fig. 1B). The central region of crystal imprints was supposed to be the site of aragonite nucleation (Rousseau et al., 2005b; Nudelman et al., 2006). After complete decalcification of lamellae, only organic interlamellar sheets remained, and individual crystal imprints on the sheets were no longer detected by a microscope (Fig. 4A). As high molecular weight of antibodies complex for immunolabeling makes them hardly diffuse through the sheets, immunolabeling of matrix proteins in decalcified nacre lamellae mostly reflects their distributions in the surface layer of interlamellar sheets (Nudelman et al., 2006). Immunolabeling experiment showed that SM proteins existed in polygonal crystal imprints in the organic matrix sheet (arrowhead) as well as the central region of the imprints (arrow, Fig. 4C), suggesting that SM proteins constitute the intertabular matrix and the nucleation sites (Nudelman et al., 2006). Immunolabeling using P-anti-NCA and M-anti-NCA both discerned polygonal crystal imprints (arrowheads), but indiscernible staining was observed in the central area (Fig. 4D and E), indicating that nacrein may be localized to intertabular matrix. Different from anti-NCA labeling, anti-P43 labeling did not outline polygonal crystal imprints, but discerned fluorescent spots widely distributing in the matrix sheet (Fig. 4E). This suggested that P43 may be in the aragonitic tablets or localized to the interlamellar sheet. In the negative control of immunolabeling, no staining was shown in the matrix sheet (Fig. 4B).
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Fig. 3. SDS–PAGE (A) and immunoblot (B) analysis of EDTA–insoluble matrix (ISM) proteins extracted from nacre of P. fucata. (A): Lane 1, SDS–extracted ISM proteins redissolved in 6 M urea; lane 2, 100 mM NaCl-eluate (E1) from a heparin affinity column; lane 3, 200 mM NaCl-eluate (E2) from the column; lane 4, the purified recombinant protein encoding the TGF-b domain of Pf-BMP-2, indicated by arrow; M, molecular weight standards, kDa. (B): Lanes 1–4 and 6, immunoblot analyzing the recombinant protein of Pf-BMP-2 (about 14 kDa), ISM proteins, E1, E2, and SM proteins (Fig. 2A, lane 1) with anti-P43 as primary antibody, respectively; lane 5, the negative control, analyzing ISM proteins with preimmune serum; arrows indicating the visualized bands.
Fig. 4. (A) Light micrograph of completely decalcified interlamellar sheets, in which only organic matrix remained; the lines in the picture are due to folds in the interlamellar sheet. (B–F) fluorescence micrographs of decalcified interlamellar sheets labeled by preimmune serum, anti-SM, P-anti-NCA, M-anti-NCA and anti-P43, respectively. Arrowheads indicate the stained intertabular matrix. Arrow in (C) indicates the stained area in the center of the crystal imprints. Bar, 20 lm.
3.4. Immunogold labeling of matrix proteins in partially etched nacre lamellae Immunogold labeling partially etched nacre lamellae will reveal the distributions of matrix proteins in aragonitic tablets, which is observed by a SEM in back-scattered electron mode (SEM-BSE). In this model, elements with higher atomic number scatter more electrons thus appearing lighter. Therefore, immunogold particles are displayed as the brightest tiny spots; the mineral phase is lighter than the organic phase. Fig. 5C and D (arrow indicating) revealed that nacrein actually distributes in intertabular matrix. Figs. 5C and D and 6C and D (arrowhead indicating) showed that this protein also widely exists in the aragonitic tablets and seems to attach to the mineral phase. As an ISM, P43 is rarely localized to the intertabular matrix (Fig. 5E and F), but exists in aragonitic tablets (arrowhead indicating, Figs. 5E and F and 6E and F). The negative control (Figs. 5A and B and 6A and B) with preimmune serum suggested that the positive staining is reliable. Fig. 7 showed the distributions of nacrein and P43 in the crosssection of nacre lamellae (section parallel to the c-axis). The immunogold labeling confirmed that nacrein widely distributes in nacre lamellae and is embedded in the mineral phase as calcium carbon-
ate crystals growing (Fig. 7C and D). P43 distributes within the lamellae (Fig. 7E and F, black arrows), and also is localized to interlamellar matrix at interface with the mineral phase (Fig. 7E and F, white arrows). As expected, the negative controls displayed a low background, which make the labeling reliable (Fig. 7A and B). 3.5. In vivo investigation of the actions of nacrein and P43 in the growth of aragonitic tablets There is a space between nacre and mantle tissue, filled with body fluid. Matrix proteins secreted by mantle tissue are released into this space and act in the growth of nacre lamellae. The purified antibodies against P43 and nacrein were injected into this space to disrupt the functions of these proteins in nacre growth. Each antibody-injected group contained four oysters. As a control, preimmune rabbit serum was injected at same dosage. Three days later, the oysters were sacrificed. The inner surfaces of the nacre were examined by a SEM. 3.5.1. Treatment of anti-P43 In low dosage anti-P43 injected-group, the aragonite nanograins failed to pack into a compact aggregation as the untreated ones (Fig. 8C), and became disordered (Fig.8C and D). As the injection dos-
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Fig. 5. Immunogold labeling matrix proteins in the intertabular matrix. (A, C, and E) Labeling the aragonitic tablets with preimmune serum, P-anti-NCA, and anti-P43, respectively. (B, D, and E) SEM-BSE observing immunogold staining in the boxes of (A, C, and E) respectively; arrows indicating immunogold particles localized to the intertabular matrix; arrowheads indicating the gold particles on the aragonitic tablets. Bars, (A–F) 2 lm, 400 nM, 3 lm, 500 nm, 3 lm, 400 nm, respectively.
Fig. 6. Immunogold labeling matrix proteins within partially etched aragonitic tablets. (A, C, and E) labeling the aragonitic tablets with preimmune serum, P-anti-NCA, and anti-P43, respectively. (B, D, and E) SEM-BSE imaging immunogold staining in the boxes of (A, C, and E) respectively; arrowheads indicating immunogold particles distributing in the aragonitic tablets. Bars, (A–F) 3 lm, 500 nM, 3 lm, 500 nm, 4 lm, 400 nm, respectively.
age doubled, the growth of aragonitic tablets was seriously disrupted. Calcium carbonate crystals with organic substance failed to assemble into aragonitic tablets within 4 4 mm2 area around the injection site (Fig. 8E and F). In contrast, aragonitic tablets of preimmune serum treated group kept the normal shape and framework (Figs. 8A and B and 9D–F). These observations indicated that antiP43 treatment disrupted the organo-mineral assemblage of aragonitic tablets, suggesting that P43 should be a framework matrix. 3.5.2. Treatment of anti-NCA in high dosage Although the aragonitic tablets appeared coarser by preimmune serum treatment than untreated ones (Fig. 9A–C), the thickness had little change (Fig. 9D–F). In contrast, P-anti-NCA treatment resulted in thickening of aragonitic tablets (Fig. 9G–I). While the normal tablets displayed uniformed surface (Fig. 9B and C), aberrant tablets did not. The surface of aberrant tablets was characterized as an accumulation of inconsistent nanograins (Fig. 9H and I). Moreover, calcium carbonate crystals precipitated randomly out-
side the aberrant aragonitic tablets. In about 4 4 mm2 area around injection sites, aberrant growth of aragonite crystal occurred in all 4 experimental oysters. Similarly, aberrant aragonitic tablets, which thickened, were also observed in the inner lamellae of M-anti-NCA injected group (Fig. 9J–L). The aberrant tablets exhibited inconsistent nanograins. Less calcium carbonate crystals were deposited around aragonitic tablets in M-anti-NCA treated group (Fig. 9K) than P-anti-NCA treated group (Fig. 9H). It is noticeable that aragonitic tablets in anti-NCA injected groups had normal polygonal shape and framework. These observations suggested that nacrein mostly functions in the regulation of the growth of calcium carbonate crystals.
4. Discussion In this study, we three-dimensionally map the distributions of SM proteins and an ISM protein in nacre lamellae, and conduct
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Fig. 7. Immunogold labeling the cross-section of partially etched nacre lamellae (section parallel to the c-axis). (A, C, and E) The lamellae labeled by preimmune serum, Panti-NCA, and anti-P43, respectively; (B, D, and F) SEM-BSE imaging the immunogold staining in the lamellae of (A, C, and E) respectively. Bars, (A and B) 500 nm; (C and D) 400 nm; (E and F) 500 nm.
Fig. 8. (A) SEM image of innermost lamella in low dosage preimmune serum treated group; (B) enlargement of the box in (A); (C) SEM image of innermost lamella in low dosage anti-P43 treated group; (D) enlargement of the box in (C) showing defective aragonitic tablets; (E) SEM image of innermost lamella in high dosage anti-P43 treated group; (F) enlargement of the box in (E) showing calcium carbonate crystals adhering with organic substance. Bars, (A, C, and E) 20 lm; (B, D, and F) 2 lm.
antibody inhibition assay to study their functions in nacre calcification. The understanding of the localization of matrix proteins is central to understanding their functions in the growth of nacre lamellae. It is known that SM proteins precisely regulate nacre mineralization, therefore control crystal morphology, crystal phase switching and orientation (Mann et al., 1993; Belcher et al., 1996; Levi et al., 1998). Herein, our immunolabeling shows that SM proteins are localized to the central region and the periphery of polygonal crystal imprints in interlamellar sheets (Fig. 4C), suggesting that SM proteins are localized to the nucleation sites of aragonitic tablets and the intertabular matrix (Crenshaw and Ristedt, 1976; Rousseau et al., 2005b; Nudelman et al., 2006). As a major component in SM proteins, nacrein is also demonstrated its distribution in the intertabular matrix by anti-NCA labeling (Figs. 4D and E and 5C and D). Since intertabular matrix associated proteins are
thought to function in the inhibition or cessation of crystal growth (Marin et al., 2000; Nudelman et al., 2006), the distribution of nacrein in intertabular matrix might suggest its function in the inhibition or cessation of crystal growth. By calcium carbonate precipitation assay, Miyamoto et al. (2005) also suggested that nacrein might act as a negative regulator in shell calcification. To learn the actual role of nacrein in the growth of nacre lamellae, we conducted antibody inhibition assays using rabbit polyclonal antibodies and a mouse monoclonal antibody against the CA domain of nacrein. A CA-dependent role of nacrein in the regulation of calcium carbonate crystal growth is revealed. In both antibodies treatment groups, the most obvious change is the thickening of aragonitic tablets resulting from disordered nanograins growth, which is distinct from the normal ones assembled by coherent nanograins with consistent crystal orientation (Rousseau et al., 2005a). Considering its wide distribution in aragonitic
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Fig. 9. (A–C) SEM images of untreated aragonitic tablets;(D–F) SEM images of innermost lamella in high dosage preimmune serum treated group; (G–I) SEM images of innermost lamella in high dosage P-anti-NCA treated group; (J–L) SEM images of innermost lamella in high dosage M-anti-NCA treated group. (B, E, H, and K) enlargement of the boxes in (A, D, G, and J) respectively. (C, F, I, and L) enlargement of the boxes in (B, E, H, and K) respectively. (C) The smooth surface of normal aragonitic tablets; (F) High dosage preimmune serum treatment resulting aragonitic tablets with coarser surface; (I and L) P-anti-NCA and M-anti-NCA treatments resulting thickening aragonitic tablets with inconsistent nanograins. Bars, (A, D, G, and J) 50 lm; (B, E, H, and K) 5 lm; (C, F, I, and L) 1 lm.
tablets (Fig. 7C and D), we believe that nacrein with CA activity likely functions as a negative regulator in the growth of nanogains in aragonitic tablets, ensuring their consistent crystal orientation and growth rate. Since CA catalyzes the reversible hydration of CO2 into HCO3 (Tripp et al., 2001), nacrein might exert its regulatory function via controlling carbonate/bicarbonate equilibrium for calcium carbonate precipitation or adjusting pH in the microenvironment of calcification. Furthermore, nacrein widely distributes over the surface of aragonitic tablets (Fig. 6C and D). This distribution allows nacrein to act as an inhibiting surface, constraining crystals growth in the c-axis direction. Therefore, as nacrein is suppressed in vivo, crystal overgrows in the c-axis direction (Fig. 9H and K). Moreover, we notice that more undesirable crystals form around aragonitic tablets after P-anti-NCA treatment (Fig. 9H) than after M-anti-NCA treatment (Fig. 9K). This could be due to the fact that more epitopes in CA domain are recognized and blocked by polyclonal antibodies than by a monoclonal antibody. These observations indicate that the negative regulation would also prevent calcium carbonate crystallizing in undesirable sites. As an ISM protein, the distribution of P43 is quite different from SM proteins. In completely decalcified lamellae, anti-P43 label is
not detectable in the periphery of crystal imprints as does antiSM, but is discerned as many fluorescent spots on the organic sheet (Fig. 4F). This suggests that P43 might be absorbed in the interlamellar sheet. In the cross-section of nacre lamellae, the immunogold labeling reveals that P43 is actually localized to interlamellar matrix sheet (Fig. 7E and F), and also embedded within aragonitic tablets (Figs. 6E, F and 7E and F). Antibody inhibition assay confirms a role of P43 in the construction of organic framework of nacre lamellae. When P43 was suppressed in vivo, calcium carbonate crystals adhering to fiber-like organic substances, but not normal aragonitic tablets, grew toward the innermost lamella (Fig. 8E and F). This observation shows that P43 is necessary for the buildup of aragonitic tablets. According to its distribution in aragonitic tablets, we suppose that this protein should participate in the buildup of the continuous organic matrix framework within aragonitic tablets as Rousseau et al. (2005a) observed, supporting coherent aggregation of nanograins. Its ability to bind heparin makes it possible to connect with other matrix within aragonitic tablets and the interlamellar matrix, for instance chitin, silk fibroin and some proteoglycans (Ruppert et al., 1996; Levi-Kalisman et al., 2001; Addadi et al., 2006).
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Since the discovery that nacre powder can induce bone formation in vivo (Westbroek and Marin, 1998), BMP homologs are hypothesized to exist in the matrix of the nacreous shell layer. P43, which is a matrix protein, can be specifically recognized by the antibodies against the recombinant protein encoding the TGF-b domain of Pf-BMP-2. This suggests that there are some homological epitopes expressed in P43 and TGF-b domain of PfBMP-2. Similar to mammalian BMP-2 (Ruppert et al., 1996), P43 exhibits an affinity for heparin in a heparin affinity chromatography. Immunoblot analysis and affinity chromatography reveal some features of P43. Future work will involve purification of P43. When fully characterized, P43 could be proved important not only in biomineralogy but also in medicine and evolutionary biology. Our results show that SM and ISM proteins differentially distribute in nacre lamellae and function differently in nacre mineralization. Some regulating activities of matrix proteins in the growth of aragonitic tablets would be indicated by antibody inhibition assays. The analysis of aberrant growth pattern of aragonitic tablets along with immunolocalization would further reveal the regulatory mechanisms of matrix proteins in nacre mineralization. Acknowledgments This work was financially supported by the National High Technology Research and Development Program of China (2003AA603430, 2006AA09Z413) and the Natural Science Foundation of China (30221003, 30530600). We are grateful to Dr. Yan Ma, Dana-Farber Cancer Institute, Boston, for critical discussion. References Addadi, L., Joester, D., Nudelman, F., Weiner, S., 2006. Mollusk shell formation: a source of new concepts for understanding biomineralization processes. Chem. Eur. J. 12, 980–987. Belcher, A.M., Wu, X.H., Christensen, R.J., Hansma, P.K., Stucky, G.D., Morse, D.E., 1996. Control of crystal phase switching and orientation by soluble molluscshell proteins. Nature 381, 56–58. Cho, J.W., Partin, J.S., Lennnarz, W.J., 1996. A technique for detecting matrix proteins in the crystalline spicules of the sea urchin embryo. Proc. Natl. Acad. Sci. USA 93, 1282–1286. Crenshaw, M.A., 1972. The soluble matrix from Mercenaria mercenaria shell. Biomineralization 6, 6–11. Crenshaw, M.A., Ristedt, H., 1976. The histochemical localization of reactive groups in septal nacre from Nautilus pompilius. In: Watabe, N., Wilbur, K.M. (Eds.), The Mechanisms of Mineralization in the Invertebrates and Plants. University of South Carolina Press, Colombia, pp. 355–367. Freeman, J.A., 1960. Influence of carbonic anhydrase inhibitors on shell growth of freshwater snail Physa heterostropha. Biol. Bull 118, 412–418. Gong, N., Li, Q., Huang, J., Fang, Z., Xie, L., Zhang, R., 2008. Culture of outer epithelial cells from the mantle tissue to study matrix protein secretion for biomineralization. Cell Tissue Res. doi:10.1007/s00441-008-0609-5. Lamghari, M., Almeida, M.J., Berland, S., Huet, H., Laurent, A., Milet, C., Lopez, E., 1999. Stimulation of bone marrow cells and bone formation by nacre: in vivo and in vitro studies. Bone 25 (Suppl. 2), 91S–99S. Levi, Y., Albeck, S., Brack, A., Weiner, S., Addadi, L., 1998. Control over aragonite crystal nucleation and growth: an in vitro study of biomineralization. Chem. Eur. J. 4, 389–396.
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