Role of Pif97 in Nacre Biomineralization: In Vitro Characterization of

Jun 28, 2015 - Pearl oyster Pinctada fucata is one of the best-studied molluscan species for ..... to be important for the formation of individual nac...
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Role of Pif97 in Nacre Biomineralization: In Vitro Characterization of Recombinant Pif97 as a Framework Protein for the Association of Organic−Inorganic Layers in Nacre So Yeong Bahn,† Byung Hoon Jo,‡ Byeong Hee Hwang,‡,§ Yoo Seong Choi,*,∥ and Hyung Joon Cha*,†,‡ †

School of Interdisciplinary Bioscience and Bioengineering and ‡Department of Chemical Engineering, Pohang University of Science and Technology, Pohang 790-784, Korea § Division of Bioengineering, Incheon National University, Incheon 406-772, Korea ∥ Department of Chemical Engineering, Chungnam National University, Daejeon 305-764, Korea S Supporting Information *

ABSTRACT: Nacre is the inner layer of the mollusc shell and provides exceptional toughness via its highly organized organic−inorganic composite structure. Pif is an organic matrix protein from the nacreous layer of the pearl oyster Pinctada fucata and exhibits regulatory behavior in nacre formation. Here, we investigated features of Pif97, the Nterminal of Pif, using a recombinant form of Pif97 produced in Escherichia coli. We observed that recombinant Pif97 was able to efficiently form a complex with calcium ions. Additionally, recombinant Pif97 showed both in vitro growth inhibition of thermodynamically stable calcite, stabilization of amorphous calcium carbonate, and exclusive binding affinity to metastable aragonite and chitin. These results imply the participation of Pif97 in the calcification of nacre including the association of the inorganic phase and polysaccharide template. We propose that recombinant Pif97 has inherent characteristics of the native form, which are significant for interrelating with organic matrix and inorganic calcium carbonate during nacre biomineralization.

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INTRODUCTION Biominerals such as bones, shells, and coral skeletons serve various functions, including structural support and protection of internal soft bodies in many different organisms. In particular, molluscan shells exhibit exceptional mechanical properties. These shells are mainly composed of two different calcium carbonate (CaCO3) layers known as the outer prismatic (calcite) and inner nacreous (aragonite) layers. The nacreous layer, called nacre, is iridescent and resilient, and it provides superior strength and toughness; the fracture resistance of nacreous aragonite is 3000-fold greater than pure aragonite.1,2 It has been widely accepted that the outstanding mechanical properties of nacre come from its highly organized structure. The mature aragonite crystals of nacre take the form of hexagonal tablets, and these inorganic phases extensively interact with intercalated organic matrix, exhibiting inorganic brick and organic mortar-like constituents.3,4 The formation of the organized biomineral is controlled by organic templates, and less than 5% of organic components in the dry weight of nacre lead to the structural arrangement of the unique organic− inorganic complex.5,6 The organic matrix is mainly composed of β-chitin, hydrophobic silk-like protein hydrogels, and hydrophilic acidic proteins.3 The chitin fibers are well aligned under individual © XXXX American Chemical Society

crystal tablets in nacre and constitute an insoluble two- or three-dimensional scaffold as the framework for the hexagonal tablet. However, it has been demonstrated that β-chitin itself does not modify the CaCO3 nucleation and growth due to a lack of chemical functional groups capable of strong interaction with CaCO3.3,7 Silk-like protein hydrogels such as Gly- and Alarich insoluble proteins with a β-sheet domain are components of the hydrophobic matrix between the chitin sheets, which has been suggested to inhibit uncontrolled calcite crystallization for favoring the growth of aragonite, as well as to serve as a spacefiller by prefilling the space to be mineralized.3,8 The hydrophilic acidic proteins are dispersed in the gel, and it has been shown that the acidic proteins play important roles in the nucleation and growth control of aragonite for each tablet.3,5,6 Many acidic matrix proteins containing carboxylate or sulfate reactive groups have been identified from the nacre of a number of molluscan species, and their full or partial sequences have also been determined.9 However, the proteins do not have common domains and sequence similarities, even though they have common features such as highly repetitive regions, Received: February 25, 2015 Revised: June 15, 2015

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Louis, MO, USA). The vector construct, pET23-rPif97, was confirmed by direct sequencing. Production and Purification of rPif97. E. coli BL21 (DE3) (Novagen) was transformed with the recombinant plasmid pET23rPif97 for the expression of rPif97. The recombinant cells were cultured in 500 mL of LB medium supplemented with 50 μg/mL ampicillin at 37 °C and 200 rpm in a shaking incubator. The expression of rPif97 was induced by 1 mM isopropyl-β-D thiogalactopyranoside (IPTG; Sigma-Aldrich) when the cell density (OD600) reached 0.8−1.0. After 8 h of cultivation, the cells were harvested by centrifugation at 4 °C and 4000 g for 10 min, and the cell pellet was resuspended with 45 mL of lysis buffer (50 mM NaH2PO4 and 300 mM NaCl; pH 8). The cells were disrupted using a sonic dismembrator (Sonics & Materials Inc., Newtown, CT, USA) at 30% power with 3 s pulse on and 10 s pulse off repetition cycle on ice. The lysate was fractionated by centrifugation at 4 °C and 10 000 g for 20 min. The supernatant was removed and designated as the soluble fraction. The resultant pellet was designated as the insoluble fraction, which was used for rPif97 purification. The insoluble pellet was resuspended in 5 mL of denaturing lysis buffer (100 mM NaH2PO4, 10 mM Tris, and 8 M urea; pH 8). After centrifugation at 10 000 g for 20 min, the urea-soluble supernatant was removed and mixed with Ninitrilotriacetic acid (Ni-NTA) resin (Qiagen, Germantown, MD, USA). The mixture was agitated for 1 h to allow the target protein to bind the resin. After washing the resin by wash buffer (100 mM NaH2PO4, 10 mM Tris, 8 M urea, and 20 mM imidazole; pH 8), rPif97 was eluted using elution buffer (100 mM NaH2PO4, 10 mM Tris, 8 M urea, and 200 mM imidazole; pH 8). The eluate was desalted using a PD-10 column (GE Healthcare, Cleveland, OH, USA) with 10 mM Tris buffer (pH 8) and stored at 4 °C for further analyses. Size Exclusion HPLC Analysis. The purified protein was confirmed by high performance liquid chromatography (HPLC) (Shimadzu, Tokyo, Japan) with Agilent Bio SEC-3 column (3 μm, 300 Å, 7.8 × 300 mm; Agilent, Santa Clara, CA, USA). Samples were prepared in phosphate buffer (50 mM NaH2PO4; pH 8), separated using isocratic method in phosphate buffer supplemented with 300 mM NaCl, and monitored by a photodiode array (PDA) detector at 280 nm. Calibration curve was determined using gel filtration standard (Bio-Rad, Hercules, CA, USA). Mass Spectrometric Analyses. The molecular weight of the purified rPif97 was confirmed by matrix-assisted laser desorption/ ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) (system model 4700; AB SCIEX, Framingham, MA, USA). The trypsin-digested purified rPif97 was analyzed by MALDI-TOF MS/ MS. MS/MS data were further analyzed by a computational proteomics analysis system with Mascot database search engine (Matrix Science, Boston, MA, USA) using the database from NCBInr. Amino Acid Composition Analysis. For amino acid composition analysis, purified rPif97 was dissolved in 6 M HCl supplemented with 5% (v/v) phenol. The protein was acid hydrolyzed at 156 °C for 1 h under argon, and the protein hydrolysate was sequentially washed with distilled water and methanol. The amino acid composition was then analyzed using an ion exchange column and ninhydrin-based detection system (Amino Acid Analyzer S4300; Sykam, Eresing, Germany). Extraction of Organic Matrix from Nacre. Acid-insoluble and sodium sodecyl sulfate (SDS)-soluble organic matrix of nacre (AIM), containing the Pif complex, was extracted based on a previously described method.15 In brief, the mineral phase of dried shells of P. fucata was dissolved by treatment with 1 M acetic acid, and the remaining AIM of the nacreous layer was extracted using 50 mM Tris buffer (pH 8) supplemented with 1% SDS (Sigma-Aldrich) and 10 mM dithiothreitol (DTT; Sigma-Aldrich). DTT-untreated AIM (nonreduced AIM) was separately prepared only for the Ca2+-binding assay and CaCO3 crystallization. After extraction, trichloroacetic acid (TCA) precipitation method was applied for all samples except for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE) analysis to remove chemicals. The resulting protein pellet was dissolved in 10 mM Tris buffer (pH 8) for further analysis. The final yield of collected proteins of AIM was about 0.1 mg/g-dried shell and the AIM included approximately 4% of native Pif97 (Table S1). If

intrinsically disordered domains, aggregation-prone sequences, and anionic functional groups for mineral binding.9−11 In vitro CaCO3 crystallization assays using these matrix components have been performed for a long time; however, nacre formation is not currently fully understood. Pearl oyster Pinctada fucata is one of the best-studied molluscan species for understanding the molecular basis of nacre formation. Pinctada matrix proteins such as nacrein,12 MSI60,13 N16,14 and Pif,15 which are responsible for nacre formation, have been intensively studied. In particular, Pif is regarded as a key regulator of nacre formation in P. fucata:15 in vivo immunolocalization results showed its preferred localization to be from the organic boundary to the nacreous layer. The extracted Pif complex induced the in vitro formation of uniform c-axis oriented aragonite crystals, which is comparable with that of the natural nacreous layer. The inhibition of Pif expression by RNA interference dramatically reduces biomineralization and leads to abnormal nacre growth. Moreover, homologues of Pif have also been found in other species from bivalves to gastropods,16 suggesting that Pif homologues may play important roles in nacre biomineralization of molluscs. Pif is post-translationally cleaved into Pif97 (N-terminal) and Pif80 (C-terminal). Both Pif97 and Pif80 are classified within the framework of an interlamellar protein family, which associates with the water-insoluble β-chitin-containing matrix that surrounds each aragonite tablet.10 Von Willebrand type A (VWA) and chitin-binding domains are conserved in all Pif97 homologues, whereas the Pif80 sequence that interacts with CaCO3 varies markedly among species.16 It is expected that the characteristic primary structure and conserved domains of Pif97 may work as a bridge among other organic macromolecules and inorganic aragonite. However, further characterization of Pif97 has been restricted by the difficulty of purifying Pif97 via extraction from the nacreous layers, because Pif97 has always been obtained as complexes of Pif97, Pif80, N16, and other proteins.15 In the present work, we considered a recombinant protein approach as a solution for obtaining individual Pif97 to analyze the role of native Pif97 in nacre biomineralization. Recombinant Pif97 (rPif97) protein was designed and successfully massproduced in a bacterial system. The features of rPif97 interactions with calcium ions (Ca2+), the CaCO3 mineral phase, and chitin were examined. We also performed in vitro CaCO3 mineralization using rPif97 in comparison with organic matrix extracted from the nacre of P. fucata.

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EXPERIMENTAL SECTION

Vector Construction. The protein sequence information on P. fucata Pif97 was obtained from Suzuki’s report.15 The gene encoding Pif97 was redesigned based on E. coli codon preference and avoidance of the repetition of major codons and chemically synthesized with the addition of N-terminal NdeI and C-terminal XhoI restriction sites (GenScript USA Inc., Piscataway, NJ, USA). The target gene was introduced into a pET-23b(+) vector (Novagen, Darmstadt, Germany) under the control of a strong T7 promoter, resulting in pET23-rPif97. The amino acid sequence of recombinant protein is expected to be identical to that of authentic Pif97, except for the additional N-terminal methionine (start codon) and eight amino acid sequences at the C terminus (LEHHHHHH; two from an XhoIrestriction site and six from the following hexahistidine (His6)-tag). E. coli TOP10 (Invitrogen, Carlsbad, CA, USA) was used as a host strain for vector construction. The cells were cultured in Luria−Bertani (LB) medium supplemented with 50 μg/mL ampicillin (Sigma-Aldrich, St. B

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Figure 1. (a) SDS-PAGE and (b) Western blot analyses of rPif97 expression in E. coli. (c) Coomassie and (d) Stains-all staining analyses of purified rPif97. Lanes: M, protein molecular weight marker; W, whole cell; S, soluble fraction; I, insoluble fraction; rPif97, purified rPif97; AIM, acidinsoluble and SDS-soluble organic matrix; BSA, bovine serum albumin. The white and black arrows indicate rPif97 and native Pif97, respectively. sonicated in isopropanol for 10 min. After washing with distilled water, they were placed at the bottom of a 24-well plate (SPL Life Science) before starting in vitro crystallization. The crystallization assays were performed at 20 or 4 °C with preincubated ingredients at assay temperatures. The assay solutions were prepared with different concentrations of protein (0, 2, 10, 50, 250 μg/mL) supplemented with 10 mM CaCl2. After 5 min, centrifugation was performed at 13 000 rpm for 3 min. Aliquots (500 μL each) of supernatant of each assay solution were placed in wells containing a coverslip. The plate was sealed with plastic wrap with a pinhole for each well, and placed in a desiccator with 0.7 g of solid (NH4)2CO3 (Sigma-Aldrich). After crystallization, glass coverslips with precipitated CaCO3 particles were rinsed with distilled water and dried at room temperature. X-ray diffraction (XRD) patterns of precipitated CaCO3 were analyzed and recorded by powder X-ray diffractometer D/Max-2500/PC (Rigaku, Tokyo, Japan) with Cu Kα radiation. Scanning was performed in the 2θ range 20−50° with 0.02° of sampling width. For morphology observations, CaCO3 particles were coated with a thin layer of Pt and imaged by field emission-scanning electron microscope (FE-SEM; XL30S FEG, Philips Electron Optics B.V., Eindhoven, Netherlands) at an accelerating voltage of 5 kV. Transmission electron microscope images and corresponding electron diffraction patterns were obtained by high-resolution transmission electron microscopy (HR-TEM) (JEOL JEM 2200FS; JEOL, Tokyo, Japan). CaCO3-Binding Assay. Calcite powder (Sigma-Aldrich) was used as supplied. Aragonite powder was manufactured according to previous studies with modifications.19,20 In brief, 50 mM calcium hydroxide powder (Sigma-Aldrich) was suspended in 150 mM magnesium chloride (Sigma-Aldrich) solution by vigorous stirring, and the mixture was heated inside a water bath. When the temperature was increased to 70 °C, CO2 gas was bubbled through a glass dispersion tube at a flow rate of 1 L/min until the pH of the mixture became constant. The resulting solution was filtered through a 0.2 μm filter membrane (Merck Millipore), and the remaining slurry was washed with ethanol (Merck Millipore) and then with distilled water. After drying the powder on the membrane at 120 °C for 2 h, the crystal phase of the aragonite was confirmed by XRD patterns and SEM images. Lysozyme (Bio Basic) and trypsin (Sigma-Aldrich) were used as control proteins in the assay. The samples were prepared by dissolving protein powders in 10 mM Tris buffer (pH 8). For the binding assay, 50 μL of each protein solution (0.5 g/L) was mixed with 10 mg of CaCO3 powder, and the mixture was incubated at 20 °C for 16 h with vigorous shaking at 220 rpm. After centrifugation at 13 000 rpm for 1 min, the supernatant was removed, and designated flowthrough (FT) and the CaCO3 pellet was sequentially washed with 50 μL of 10 mM Tris buffer (pH 8) (W1) and the same buffers with 0.1 M NaCl (W2) and 0.5 M NaCl (W3). Finally, the remaining CaCO3 crystals were dissolved in the same volume of 4 M acetic acid (E).

necessary, the extracted proteins were concentrated by ultrafiltration (Amicon Ultra-0.5; EMD Millipore, Billerica, MD, USA). SDS-PAGE and Western Blot Analyses. For SDS-PAGE analysis, each sample was mixed with sample buffer (50 mM Tris, 2% SDS, 10% glycerol, 1% β-mercaptoethanol, and 0.004% bromophenol blue; pH 6.8), and boiled at 100 °C for 10 min. Bovine serum albumin (BSA; Promega, Madison, WI, USA) was used as a control protein. Proteins were separated by 12% SDS-polyacrylamide gel according to the standard protocol and stained with Coomassie blue R-250 (Bio-Rad). The intensity of the band of interest was evaluated by Gel-Pro analyzer software (Media Cybernetics, Silver spring, MD, USA). For Western blot analysis, the proteins separated on the gel were transferred to a nitrocellulose membrane (Thermo Fisher Scientific, Hampton, NH, USA). After sequential incubation with a primary monoclonal anti-His6 antibody (abm, New York, NY, USA) and a secondary anti-mouse IgG alkaline phosphataseconjugated antibody (Sigma-Aldrich), the target protein was visualized by a color development reaction using nitro blue tetrazolium (NBT; Bio Basic, Amherst, NY, USA) and 5-bromo-4-chloro-3-indolyl phosphate (BCIP; Bio Basic). Gel Staining Assays. Stains-all staining was conducted based on the previously described method.17 The gel after SDS-PAGE was washed exhaustively twice in 25% isopropanol (Duksan, Ansan, Korea) to remove SDS, and fixed overnight with 25% isopropanol. The gel was soaked in fresh Stains-all solution of 0.005% Stains-all (SigmaAldrich), 10% formamide (Merck Millipore, Darmstadt, Germany), 25% isopropanol, and 15 mM Tris (pH 8.8) and stained for 24 h in dark conditions at room temperature with gentle agitation. After being washed in distilled water several times, the gel was scanned using a gel scanner. In addition, Periodic acid-Schiff (PAS) staining (Thermo Fisher Scientific) and Pro-Q Diamond staining (Invitrogen) were performed on the proteins of AIM after SDS-PAGE according to the manufacturer’s instructions to identify glycosylation and phosphorylation of the proteins. Turbidimetric Measurement of Ca2+-Induced Protein Agglomeration. Turbidimetry was performed based on the previously described method18 with the following modifications. To analyze agglomeration in different Ca2+ concentrations, a range of CaCl2 (Sigma-Aldrich) aqueous solution was directly mixed with a 0.3 g/L protein solution at a ratio of 1:9 (v/v). After incubation for 5 min at room temperature, the samples were transferred to a flat-bottom, 96well plate (SPL Life Science, Pocheon, Korea) and the absorbance was measured at 600 nm using a microplate spectrophotometer (Bio-Rad). The experiments were performed in triplicate. In Vitro CaCO3 Crystallization. The effect of rPif97 on in vitro CaCO3 crystallization was investigated by exploiting slow diffusion of ammonium carbonate vapor in calcium chloride solution.5,6 Glass coverslips (Marienfeld-Superior, Lauda-Königshofen, Germany) were C

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Protein contents were analyzed by dot blotting on a nitrocellulose membrane, followed by Coomassie blue staining. Chitin-Binding Assay. The chitin-binding assay was performed as described21 with slight modifications. Proteins were incubated with 2 g/L chitin derived from shrimp (Sigma-Aldrich) in 500 μL of 8 mM Tris (pH 8) at 25 °C for 4 h with shaking at 220 rpm. The supernatant (FT) was removed after centrifugation at 13 000 rpm for 3 min. The remaining chitin pellet was first washed with 500 μL fresh 10 mM Tris buffer (pH 8) (W1) and then washed with the same buffer supplemented with 0.1 M NaCl (W2). The protein content of each fraction was analyzed by SDS-PAGE followed by silver staining (BioRad).

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RESULTS Preparation of rPif97 Protein. rPif97 protein was expressed as an insoluble form in E. coli, and the band corresponding to the protein in both SDS-PAGE and Western blot analyses agreed well with that of the native Pif97 of AIM from P. fucata (Figure 1a−d). The target rPif97 was successfully purified by His6-tag affinity chromatography under denaturing conditions which was confirmed by HPLC analysis (Figure S1). The recombinant protein was prepared with a purity of approximately 90% determined by gel image analysis (Figure 1c) and a final yield of 4 mg/L-culture (Table S1). The molecular weight of rPif97 determined by MALDI-TOF MS was 59.8 kDa (Figure S2), which was almost identical to the theoretical molecular weight (60 kDa). The purified rPif97 also showed an amino acid content that is almost the same with the theoretical amino acid composition of rPif97 (Figure S3). Additionally, tryptic peptides from the purified rPif97 were analyzed by MALDI-TOF MS/MS and the identity of the purified rPif97 was confirmed (Figure S4). Through PAS and Pro-Q Diamond stainings, we found that the native Pif97 of AIM was not glycosylated and phosphorylated, respectively (data not shown), although other post-translational modifications (PTMs) may exist. The native Pif97 and purified rPif97 appeared in similar locations with a size of approximately 70 kDa on SDS-PAGE (Figure 1d), although the molecular weight of rPif97 was 59.8 kDa. The unusually acidic properties (the calculated isoelectric point (pI) value was 4.9) of Pif97 might result in slower mobility than expected due to negative charge repulsion with SDS.22 Ca2+-Binding of rPif97. Interaction with Ca2+ is an important characteristic of calcification-related proteins. Thus, the possible Ca2+-binding ability of rPif97 was first investigated by Stains-all staining, which is a commonly used analytical method for Ca2+-binding proteins.17 While BSA, which is also an acidic protein with a pI value of 4.7, which is similar to that of rPif97,23 was stained pink (negative staining), both rPif97 and native Pif97 of AIM were visualized as blue in color (positive staining) (Figure 1d). It is known that anionic sites such as sialic acid or phosphoryl groups from PTMs can be stained blue.17 However, because rPif97 does not contain any PTMs due to the intrinsic inability of E. coli, the positive staining of rPif97 by Stains-all clearly indicates that the potential Ca2+-binding capacity of Pif97 seems to imply its primary structure, particularly carboxylated acidic amino acid clusters. Next, we investigated the direct interaction of rPif97 with Ca2+ by simple mixing. Interestingly, the addition of Ca2+ induced agglomeration of rPif97 (Figure 2a). These agglomerates were not reversibly dissolved in calcium deficient buffer, but were immediately dissolved upon the treatment of ethylenediaminetetraacetic acid (EDTA) (Figure 2a). This

Figure 2. (a) Photographs of Ca2+-induced agglomeration of rPif97: (i) rPif97 solution in 10 mM Tris (pH 8), (ii) addition of 10 mM CaCl2, (iii) centrifugation at 16 000 g for 15 min, (iv) washing with 10 mM Tris (pH 8) and centrifugation, and (v) addition of 10 mM EDTA. (b) Turbidimetric measurement of Ca2+-induced agglomeration. rPif97, purified rPif97; AIM, acid-insoluble and SDS-soluble organic matrix; BSA, bovine serum albumin.

phenomenon indicates that the agglomerates are complexes of protein and Ca2+. The extent of agglomeration was changed according to Ca2+ concentration, which was measured by turbidimetry (Figure 2b). While Ca2+ did not induce the agglomeration of BSA, both dithiothreitol (DTT)-reduced and nonreduced AIMs (including native Pif-complex) showed similar degrees of Ca2+-induced agglomeration compared to rPif97. The higher turbidity of reduced AIM compared with that of nonreduced AIM may be due to a greater number of exposed sites for calcium interaction by DTT-induced release of Ca2+-interacting proteins from the disulfide-linked protein complexes of AIM. While both AIM samples had broad ranges of Ca2+ concentration at which the agglomeration occurred, rPif97 predominantly agglomerated at low Ca2+ concentration near 10 mM, which is similar to conditions in natural seawater (∼10.5 mM).24 Effect of rPif97 on In Vitro CaCO3 Crystallization. The effect of rPif97 on the crystallization of CaCO3 was investigated using the ammonium carbonate vaporization method. Nonreduced AIM (including native Pif-complex) and BSA were used as positive and negative controls, respectively. It is widely known that thermodynamically stable calcite is spontaneously formed in vitro under ambient conditions. As expected, standard rhombohedra of calcites were mainly formed during a 16 h incubation at 20 °C when no protein was added (Figure 3a). BSA also resulted in calcite crystals, confirmed by XRD, without morphological defects throughout the entire range of concentrations examined (0−250 μg/mL) (Figures 3b and S5). These results are consistent with those of a previous study.25 In contrast to BSA, polycrystalline calcites were shown in the D

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rhombohedra together with Pif97, consequently producing anhedral crystals. Mineralization experiments were also performed at 4 °C to efficiently investigate the effect of rPif97 on in vitro CaCO3 crystallization by slowing down nucleation and crystal growth. While standard rhombohedra of calcite were also formed during a 16 h incubation at 4 °C without addition of protein (Figure 4a), similar growth inhibition of calcite was observed in both SEM and XRD analyses in the presence of rPif97 (Figure 4b−d). Interestingly, we observed some intriguing features; the surfaces of specimens were covered with smaller deposits beside the CaCO3 precipitates (inset, Figure 4b). These deposits were gradually discerned according to the added concentration of rPif97 (Figure S6); they covered more than half of the specimen with the addition of 50 μg/mL, and almost the entire area with 250 μg/mL. The deposits were recognized as two types; micrometer-level deposits (white arrow, Figure 4b) and submicrometer-level deposits (black arrow, Figure 4b). The former deposits were observed on the entire surface of the specimen and were present even when ammonium carbonate vapor was not supplied for in vitro crystallization (data not shown). Thus, the agglomerates might have been formed by redundant reaction of Ca2+ and rPif97. However, the latter deposits were only observed in the in vitro CaCO 3 crystallization via ammonium carbonate vapor, which indicates they are not just a rPif97 superstructure. The deposits notably generated the “inhibition zone” region of grown CaCO3, and they gradually disappeared with increased incubation time (Figure 4b−d). TEM images showed that these submicrometer-level deposits were particles with irregular shapes (Figure 4e) with an amorphous phase, showing no electron diffraction pattern (Figure 4f). Therefore, we assumed that the initially generated inhibition zone might be formed by consumption of the submicrometer-level deposits, as a form of amorphous CaCO3 (ACC), to support the growth of CaCO3. Such an ACC stabilization capability of rPif97 might help to control biomineralization by inhibiting the transition from the ACC phase to the crystalline phase. Although consumption of ACC as a component of other crystalline CaCO3 was not verified in this work, spherulitic precipitates (inset, Figure 4d) observed after long incubation times (48 h) seemed to be newly produced CaCO3 compared to growth-inhibited calcite precipitates (Figure 4b,c). Binding of rPif97 to CaCO3 Mineral Phase and Chitin. Framework or interlamellar proteins, including Pif, are located in the area surrounding each inorganic aragonite tablet and βchitin.10 Therefore, the interactions of rPif97 with CaCO3 minerals and chitin were examined. Purified rPif97 was mixed with calcite or aragonite polymorph of CaCO3, and the binding capability was examined by dot-blotting. Lysozyme, trypsin, and AIM were used as control proteins. Because none of the tested proteins bound to calcite, they were all recovered in the flowthrough (FT) and first-wash (W1) fractions (Figure 5a). However, AIM and rPif97 exhibited effective binding to aragonite, and thus were not consequently recovered in FT, W1, or even after washing with high-salt buffer (W2−W3). The proteins were released only when the protein-bound aragonite was dissolved by acetic acid (E). Because the C-terminal his6tag of rPif97 was not responsible for binding to aragonite, which was verified using other his6-tagged proteins (data not shown), the aragonite-binding could be regarded as an inherent function of Pif97. In addition, because one of the distinctive structural characteristics of Pif97 is the existence of a putative

Figure 3. SEM images (upper) and XRD spectra (lower) of CaCO3 precipitates obtained at 20 °C for 16 h incubation without additive (a) and in the presence of BSA (b), AIM (c), and rPif97 (d). Concentrations of additive proteins were all 250 μg/mL. The insets are magnified images. Peaks indicated by asterisks correspond to calcite phase of the XRD spectra.

presence of 50 μg/mL of AIM, and the morphology was drastically changed to a rounded form when 250 μg/mL of AIM was added, which seemed to severely inhibit growth (Figures 3c and S5). The precipitated CaCO3 crystals influenced by rPif97 conspicuously featured polycrystalline calcite when 2 μg/mL of the protein was added (Figure S5). Stair-like pits and truncations of corners and edges of rhombohedra were gradually more observed with the increase of rPif97 concentration (Figures 3d and S5). These truncated morphologies seemed to be the result of inhibiting the growth of specific crystal planes of calcite.26,27 The calculated molar concentration of 250 μg/mL of rPif97 corresponds to ∼4.2 μM, which is comparable to the concentrations commonly used in in vitro CaCO3 crystallization assay using peptides or lowmolecular-weight proteins.28−32 Note that even the lowest concentration (2 μg/mL) of rPif97 affected growth of CaCO3, which showed polycrystalline morphology (Figures S5 and S6). The morphological defects of AIM and rPif97 were accompanied by a decrease in crystallinity, as shown in XRD analyses (Figure 3c,d). Other organic components in AIM might also inhibit the growth of different crystal planes of E

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Figure 4. SEM images (upper, a−d), TEM image (upper, e), and accompanying electron diffraction pattern (upper, f), and XRD spectra (lower) of CaCO3 precipitates obtained at 4 °C without additive (a) for 16 h incubation and in the presence of rPif97 for 16 h (b), 24 h (c), and 48 h (d) incubation. The concentration of rPif97 was 250 μg/mL. The insets in panels (b) and (d) are magnified images. The white and black arrows in the inset of image (b) indicate micrometer-level deposits and submicrometer-level deposits, respectively. Deposits indicated by the black arrow in the inset of (b) were analyzed by TEM. Peaks indicated by asterisks correspond to the calcite phase of the XRD spectra.

chitin-binding domain,15,16 the binding of rPif97 to chitin was experimentally confirmed using a chitin-binding assay. As expected, rPif97 was tightly bound to chitin (∼99%) and was not washed out, while BSA showed no detectable binding to chitin (Figure 5b). The chitin-bound rPif97 was released only by boiling with SDS (data not shown).

nucleation of individual aragonite tablets, and (4) growth of the tablets to form the mature tissue.3 In the first stage, mantle cells produce macromolecules including Pif97 and concentrate Ca2+ and CO32−/HCO3− ions for mineralization. Matrix organic macromolecules bind Ca2+ with stoichiometries that often exceed the number of potential anionic binding sites, which induces crystal nucleation.33 Our study showed that rPif97 strongly bound Ca2+ and induced rapid agglomeration. Thus, Pif97 expressed in mantle epithelium,15 as a component of AIM, is expected to efficiently complex Ca2+ from the marine environment and store Ca2+. Because Ca2+-induced agglomeration has also been shown in other calcification-related proteins, such as osteonectin18 and phosphophoryn34 from bone and dentin, this can be considered a required process for calcification. Although we did not further probe the chemical nature of the interaction and/or the Ca2+-binding site on rPif97, protein−calcium interaction is regarded as an important event for the initial mineral formation and the consequent regulation of calcification. Based on these results, the proposed functional roles of Pif97 are illustrated in Figure 6. It has been suggested that the initial mineral phase of ACC is formed in mantle vesicles and then delivered to the mineralization site.3 The CaCO3 supplied by direct deposition from CaCO3 solution is logistically insufficient for achieving the necessary aragonite tablet volume, and the mineral-containing

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DISCUSSION Here, the role of Pif97 in the nacre biomineralization of bivalves was analyzed based on the biochemical properties of rPif97. The intrinsic disorder propensity of Pif9710 and the functional contribution of denatured Pif complex to oriented aragonite crystallization15 suggest that an exact three-dimensional structure derived from the natural folding system of P. fucata is not crucial for the function of Pif97 as a key regulator of nacre formation. Considering the limited structural requirements for its proper function and relatively few possible PTMs in Pif97, the rPif97 produced in E. coli without PTMs is also expected to have functional roles comparable to the native protein. Although the assembly of organic matrix macromolecules and interactions of the organic templates with the inorganic phase have not been revealed in detail, the biomineralization mechanism of nacre has been previously proposed as consisting of the following four stages: (1) assembly of the matrix, (2) the first-formed mineral phase, (3) F

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ACC and inhibition of calcite growth achieved by Pif97 might be required processes for inducing nacreous aragonite formation. Similar restriction was also conducted by the ACC-binding protein of P. fucata.42 With its macromolecule interaction sites, Pif97 may prevent unwanted crystallization at desired locations for proper nucleation of aragonite, which can also be performed by other proteins, such as Pif80 and N16. β-Chitin not only is the major organic component of interlamellar sheets of nacre, but is also considered as a possible nucleation site for individual nacreous aragonite tablets through functional interactions with proteins.3,28,43 From the properties of rPif97 related to chitin and the mineral phase, we assume that rPif97 has a role in arranging the CaCO3 inorganic phase on the chitin surface. In addition, the VWA domain of Pif97 may be involved in the interaction with organic matrices, including silk-like protein hydrogels.15,16 Although both rPif97 and AIM inhibited calcite crystal growth, perceivable formation of nacreous aragonite was not observed under our experimental conditions. The formation of oriented aragonite crystals was previously demonstrated on chitin surfaces using an extracted Pif-complex of AIM.15 Silk-like protein hydrogel as well as chitin could be one of the crucial components for the proper function of AIM macromolecules, including Pif97, in aragonite formation.7,8 Nevertheless, the growth inhibition of thermodynamically favorable calcite and stabilization of unstable ACC by rPif97 shown in this study have significant implications; these can be regarded as the prerequisite processes for producing the less favorable phase of CaCO3 such as aragonite. Moreover, the effective binding affinity of rPif97 for aragonite suggests the feasibility of its participation in aragonite mineralization.

Figure 5. (a) Dot-blotting with Coomassie staining after the CaCO3 binding assay. Lanes: FT, flowthrough fraction; W1, wash fraction with 10 mM Tris (pH 8); W2, wash fraction with 10 mM Tris (pH 8) supplemented with 0.1 M NaCl; W3, wash fraction with 10 mM Tris (pH 8) supplemented with 0.5 M NaCl; E, dissolved CaCO3 with 4 M acetic acid. (b) Chitin-binding assay. Lanes: Ctrl, sample before binding assay; FT, flowthrough fraction; W1, wash fraction with 10 mM Tris (pH 8); W2, wash fraction with 10 mM Tris (pH 8) supplemented 0.1 M NaCl.

granules in the epithelial cells of the mantle are also composed of ACC. Because ACC in its pure form is highly unstable and thus rapidly transforms to the crystalline phase, ACC in a biological system is stabilized by specialized macromolecules such as acidic proteins and polysaccharides.35 These biogenic ACCs exist in a wide range of living organisms, including molluscs,36 sea urchins,37 corals,38 and crustaceans.39 Importantly, the transient amorphous phase has been suggested as a precursor for the crystalline CaCO3 phase of mollusc shells.3,40 Moreover, phase transformation from ACC or metastable vaterite to stable aragonite is hypothesized to be important for the formation of individual nacre tablets.41 Thus, stabilization of

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CONCLUSIONS In summary, we investigated functional features of Pif97 using rPif97, which was prepared using a recombinant E. coli expression system, to understand its role in nacre biomineralization. rPif97 efficiently formed a complex with Ca2+ and showed both inhibition of calcite crystal growth and ACC stabilization in vitro, which are very important in the firstformed mineral phase. Exclusive binding of rPif97 to metastable

Figure 6. Schematic illustration of the proposed role of Pif97 in nacre biomineralization. G

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(18) Kuboki, Y.; Takita, H.; Komori, T.; Mizuno, M.; Furu-uchi, E.; Taniguchi, K. Calcif. Tissue Int. 1989, 44, 269−277. (19) Park, W. K.; Ko, S. J.; Lee, S. W.; Cho, K. H.; Ahn, J. W.; Han, C. J. Cryst. Growth 2008, 310, 2593−2601. (20) Santos, R. M.; Ceulemans, P.; Van Gerven, T. Chem. Eng. Res. Des. 2012, 90, 715−725. (21) Inoue, H.; Ozaki, N.; Nagasawa, H. Biosci., Biotechnol., Biochem. 2001, 65, 1840−1848. (22) Shirai, A.; Matsuyama, A.; Yashiroda, Y.; Hashimoto, A.; Kawamura, Y.; Arai, R.; Komatsu, Y.; Horinouchi, S.; Yoshida, M. J. Biol. Chem. 2008, 283, 10745−10752. (23) Ge, S. R.; Kojio, K.; Takahara, A.; Kajiyama, T. J. Biomater. Sci., Polym. Ed. 1998, 9, 131−150. (24) Pfeiler, E. Mar. Biol. 1997, 127, 571−578. (25) Yan, Z. G.; Jing, G.; Gong, N. P.; Li, C. Z.; Zhou, Y. J.; Xie, L. P.; Zhang, R. Q. Biomacromolecules 2007, 8, 3597−3601. (26) DeOliveira, D. B.; Laursen, R. A. J. Am. Chem. Soc. 1997, 119, 10627−10631. (27) Xu, X. R.; Pan, H. H.; Tang, R. K.; Cho, K. CrystEngComm 2011, 13, 6311−6314. (28) Keene, E. C.; Evans, J. S.; Estroff, L. A. Cryst. Growth Des. 2010, 10, 1383−1389. (29) Keene, E. C.; Evans, J. S.; Estroff, L. A. Cryst. Growth Des. 2010, 10, 5169−5175. (30) Collino, S.; Kim, I. W.; Evans, J. S. Cryst. Growth Des. 2006, 6, 839−842. (31) Ndao, M.; Keene, E.; Amos, F. F.; Rewari, G.; Ponce, C. B.; Estroff, L.; Evans, J. S. Biomacromolecules 2010, 11, 2539−2544. (32) Amos, F. F.; Destine, E.; Ponce, C. B.; Evans, J. S. Cryst. Growth Des. 2010, 10, 4211−4216. (33) Mann, S. Nature 1988, 332, 119−124. (34) Kuboki, Y.; Fujisawa, R.; Aoyama, K.; Sasaki, S. J. Dent. Res. 1979, 58, 1926−1932. (35) Addadi, L.; Raz, S.; Weiner, S. Adv. Mater. 2003, 15, 959−970. (36) Nassif, N.; Pinna, N.; Gehrke, N.; Antonietti, M.; Jager, C.; Colfen, H. Proc. Natl. Acad. Sci. U. S. A. 2005, 102, 12653−12655. (37) Politi, Y.; Arad, T.; Klein, E.; Weiner, S.; Addadi, L. Science 2004, 306, 1161−1164. (38) Meibom, A.; Cuif, J. P.; Hillion, F. O.; Constantz, B. R.; JuilletLeclerc, A.; Dauphin, Y.; Watanabe, T.; Dunbar, R. B. Geophys. Res. Lett. 2004, 31 DOI: 10.1029/2004GL021313. (39) Dillaman, R.; Hequembourg, S.; Gay, M. J. Morphol. 2005, 263, 356−374. (40) Weiner, S.; Levi-Kalisman, Y.; Raz, S.; Addadi, L. Connect. Tissue Res. 2003, 44, 214−218. (41) Qiao, L.; Feng, Q. L.; Lu, S. S. Cryst. Growth Des. 2008, 8, 1509−1514. (42) Ma, Z. J.; Huang, J.; Sun, J.; Wang, G. N.; Li, C. Z.; Xie, L. P.; Zhang, R. Q. J. Biol. Chem. 2007, 282, 23253−23263. (43) Kumagai, H.; Matsunaga, R.; Nishimura, T.; Yamamoto, Y.; Kajiyama, S.; Oaki, Y.; Akaiwa, K.; Inoue, H.; Nagasawa, H.; Tsumoto, K.; Kato, T. Faraday Discuss. 2012, 159, 483−494.

aragonite and chitin indicates that Pif97 plays an important role in the association of the inorganic phase and polysaccharide template as well as in the controlled nucleation of the initial mineral phase. Therefore, we propose that this recombinant protein has the inherent and expected characteristics of the native Pif97, which are significant for understanding nacre biomineralization mechanisms and designing novel nacreinspired organic−inorganic hybrid materials.

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ASSOCIATED CONTENT

* Supporting Information S

Table S1: Preparation of rPif97 and AIM. Figures: size exclusion HPLC chromatogram; MALDI-TOF mass spectrum of purified rPif97; amino acid composition analysis of purified rPif97; SEM images. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.cgd.5b00275.

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. Phone: +82-42-821-5682. *E-mail: [email protected]. Phone: +82-54-279-2280. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the Marine Biotechnology Program (Marine BioMaterials Research Center; to H.J.C. and Y.S.C.) funded by the Ministry of Oceans and Fisheries, Korea.

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REFERENCES

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