Effects of Peptides on CaCO3 Crystallization: Mineralization Properties of an Acidic Peptide Isolated from Exoskeleton of Crayfish and Its Derivatives Yuya Yamamoto,† Tatsuya Nishimura,† Ayae Sugawara,† Hirotaka Inoue,‡ Hiromichi Nagasawa,‡ and Takashi Kato*,†
CRYSTAL GROWTH & DESIGN 2008 VOL. 8, NO. 11 4062–4065
Department of Chemistry and Biotechnology, School of Engineering, The UniVersity of Tokyo, Hongo, Bunkyo-ku, Tokyo 113-8656, Japan and Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The UniVersity of Tokyo, Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan ReceiVed May 1, 2008; ReVised Manuscript ReceiVed July 12, 2008
ABSTRACT: Acidic proteins play key roles in biomineralization processes. Herein, we report on the effects of an acidic peptide, calcification associated peptide-1 (CAP-1), isolated from the exoskeleton of the crayfish and its derivatives on crystallization of CaCO3 on glass and chitin substrates. The morphologies of CaCO3 crystals depended on the chemical structure of the peptides. On chitin matrices, uniaxially oriented CaCO3 crystals were formed due to the effects of the specific chitin binding region of the peptides. It was revealed that the C-terminal acidic region and the 70th phosphoserine exerted great effects on CaCO3 crystallization. Introduction Biominerals are inorganic/organic composites formed by living organisms.1 Materials scientists can obtain ideas for materials design from the structures and properties of biominerals.2 Biominerals exhibit significant structural features such as elaborate morphology, controlled polymorphs, and precisely oriented crystallography which are difficult to find in simple inorganic minerals. For example, nacre of seashell is composed of layers of macromolecules and aragonite crystal tablets.3 These aragonite crystals align with their c axes perpendicular to the layer. This layered structure contributes to its high mechanical strength and pearl luster. From an early period of the research on biominerals, the existence of acidic proteins in these mineralized tissues was widely reported and the proteins were considered to play important roles in biomineralization processes. Several acidic proteins were extracted from biominerals and their in vivo and in vitro effects on crystallization were intensively examined.4 Falini et al. revealed that the fractions of acidic proteins from aragonite and calcite layer induced the formation of aragonite and calcite on β-chitin/silk fibroin composites, respectively.5 Although this result showed that these acidic proteins play key roles in the determination process of CaCO3 polymorphs, the relationship between individual proteins and their functions was still obscure because purification and characterization of these acidic proteins were very difficult due to their acidic nature. These experimental difficulties are now being solved by rapid progress of biotechnology. Some of these acidic proteins from calcified tissue such as teeth,6 bones,7 shells,8 and exoskeleton of the crustaceans9 were purified, and their amino acid sequences and nucleotide sequences of the cDNAs encoding these proteins have been determined. For example, Aspein,8h Asprich,8k and Prismalin-148f were sequenced and their in vitro effects on crystallization were examined.8g,l,n Although some acidic pro* To whom correspondence should be addressed. Fax: (+81)3-5841-8661. E-mail:
[email protected]. † Department of Chemistry and Biotechnology, School of Engineering, The University of Tokyo. ‡ Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo.
Figure 1. Amino acid sequences of CAP-1 and its recombinant peptides.
teins are being purified and sequenced, whole chemical structures including posttranslational modifications have been scarcely determined. Simple model proteins with determined chemical structures are required for further understanding of the relationship between individual acidic parts and their effects on CaCO3 mineralization. Inoue et al. isolated calcification associated peptide-1 (CAP1) from the exoskeleton of the crayfish, Procambarus clarkii.10 Amino acid sequence of CAP-1 is shown in Figure 1. The chemical structure of CAP-1 has been determined including phosphorylation of its 70th serine residue. CAP-1 consists of 78 amino acids and is rich in acidic amino acids. The C-terminal region of CAP-1 is especially acidic because of the phosphoserine at the 70th residue and an Asp-repeat. CAP-1 also has a Rebers-Riddiford (R&R) consensus sequence11 which is considered to bind R-chitin selectively. Calcium- and chitin-binding ability of the peptide has been ascertained experimentally.10 This bifunctionality indicates that CAP-1 is a mediating peptide between inorganic minerals and organic matrices in the calcified tissues. Therefore, our intention is to examine its structurefunction relationship by using recombinant peptides in three components system which we have developed to construct CaCO3/polymer hybrid thin films.12 This system consists of inorganic mineral, water-soluble additive, and water-insoluble matrix. It is well-suited to examine the mediating ability of the peptide. In our previous study,13 we prepared the uniaxially
10.1021/cg800447w CCC: $40.75 2008 American Chemical Society Published on Web 09/20/2008
Effects of Peptides on CaCO3 Crystallization
oriented calcite thin film crystals via amorphous calcium carbonate (ACC)14-16 precursor on chitin matrices in the presence of CAP-1. Recombinant peptide (rCAP-1), in which the characteristic phosphoserine residue is substituted with serine, also induced the uniaxially oriented CaCO3 crystals on chitin matrices.13 However, the surface morphology of the formed crystals is different between crystals obtained in the presence of CAP-1 and rCAP-1. Although the detailed formative mechanism of the surface structure is not fully understood, the lack of a phosphate group in rCAP-1 may decrease its ACC stabilization ability and give rise to more intensive development of the {104} calcite crystal face. These results indicate that only one site of modification can drastically affect the crystallization of CaCO3. Therefore, it is interesting to examine the effects of variations in the structure of the peptide on mineralization. Herein, we report on the structure-function relationship of CAP-1 and its related recombinant peptides. Each recombinant peptide is designed to determine the effects of certain regions of CAP-1 on CaCO3 mineralization. Experimental Section Materials. Calcium carbonate (calcite) and chitin (R-chitin from crab) were purchased from Wako (Osaka, Japan). Preparation of Recombinant Peptides. rCAP-1 and other recombinant peptides were expressed in Escherichia coli cells as reported previously.17 Preparation of Chitin Matrix. Chitin (1 wt%) was dissolved in a mixed solvent of N,N- dimethylacetamide and N-methyl-2-pyrrolidone (50/50: w/w) containing LiCl (3.0 wt%). The chitin solution was spin coated on a glass substrate. It was then washed with 2-propanol, water, and hot water (80 °C), successively. Crystallization of Calcium Carbonate. Calcium carbonate was crystallized in hanging drops of supersaturated aqueous solution of calcium carbonate using protein crystallization chambers. Preparation of the supersaturated solution was as follows: Calcium carbonate (1.8 g L-1) was suspended in water obtained from an Auto pure WT100 purification system (Yamato, relative resistivity: maximum 1.8 × 107 Ω cm). Carbon dioxide gas (99.7%) was bubbled into a stirred suspension for 3 h at 30 °C. The remaining solid CaCO3 was then removed by filtration. The concentration of calcium ion in the solution was determined by EDTA titration according to standard procedures. The [Ca2+] was made up to 6.5 mM by adding an appropriate amount of water. The glass coverslip or chitin matrix was then inverted over a well of the crystallization chamber containing 40 µL of water, which traps CO2 diffusing out of the calcium carbonate solution. The crystallization temperature was maintained at 25 °C in an incubator. Characterization. Polarizing optical microscopy images were taken with an Olympus BX51 polarizing optical microscope. Scanning electron microscopy (SEM) images were obtained using a Hitachi S-900S field-emission SEM operated at 6 kV. Samples were platinum coated using a Hitachi E-1030 ion sputter.
Results and Discussion Amino Acid Sequences of CAP-1 and Its Recombinant Peptides. Amino acid sequences of CAP-1 and its recombinant peptides are shown in Figure 1.17 CAP-1 is an acidic peptide consisting of 78 residues.10 The N- and C-terminal regions of CAP-1 are acidic. The C-terminal region is especially acidic because of the 70th phosphoserine residue and the Asp-repeat. Furthermore, CAP-1 has a R&R consensus sequence in its middle part of the sequence. rCAP-1 is a recombinant peptide of CAP-1 that lacks phosphorylation of the 70th serine. S70D is also a recombinant peptide of CAP-1 in which the phosphoserine is substituted by aspartic acid. ∆N and ∆C are recombinant peptides that lack the N- and C-terminal 17 residues, respectively. ∆N and ∆C are designed to have the same number of acidic amino acid residues. These recombinant peptides have
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Figure 2. SEM images of CaCO3 crystals grown on the glass substrates in the presence of CAP-1: (a) 0.3 × 10-3 wt%; (b) 1.0 × 10-3 wt%; (c) 3.0 × 10-3 wt% and rCAP-1; (d) 0.3 × 10-3 wt%; (e) 1.0 × 10-3 wt%; (f) 3.0 × 10-3 wt%.
the additional Ala at the N-terminus because of the technical reason on protein expression in E. coli.17 CaCO3 Crystallization on Glass Substrates. To examine the effects of CAP-1 and its recombinant peptides on CaCO3 mineralization, CaCO3 crystallization experiments were conducted on glass substrates in the presence of the peptides. Figure 2a-f shows the scanning electron microscopy (SEM) images of CaCO3 crystals grown on glass substrates in the presence of CAP-1 and rCAP-1. At a concentration of 3.0 × 10-4 wt%, rhombohedral crystals with fractured edges were obtained (Figure 2a,d). The fractured areas become larger with the increase of the concentration of the peptide (Figure 2b,e), and rounded crystals were eventually formed at a concentration of 3.0 × 10-3 wt% (Figure 2c,f). CAP-1 and rCAP-1 may be adsorbed on the growing surface of calcite crystals and affect their crystal habit. It was reported that similar calcite crystals with fractured {104} faces were formed in the presence of synthetic acid-rich polymers.18 It is worth noting that the surface morphology of the crystals formed in the presence of rCAP-1 was different from that formed in the presence of CAP-1 (Figure 2c,f). CaCO3 crystals grown in the presence of rCAP-1 consisted of larger unit crystals. The similar change in unit crystal size was observed in the crystallization on chitin matrices.13 The absence of the 70th phosphate group may decrease the inhibitory abilities of the peptide for crystallization. Importance of the phosphate group can be seen in the case of Statherin, an acidic peptide from human saliva. Statherin also has a phosphoserine residue and acidic sequence in the N-terminal region, and this region is thought to interact with hydroxyapatite.6c For further examination of the effects of the acidic C-terminal region of the peptide on the crystallization, we used recombinant peptides: S70D, ∆N, and ∆C (Figure 1). CaCO3 crystals formed on glass substrates in the presence of these peptides as shown in the SEM images (Figure 3). First, we used S70D, which is a recombinant peptide of CAP-1 with its 70th phosphoserine substituted to aspartic acid. It was not considered that a conformational change was caused by the substitution of the 70th residue because the CD spectra of CAP-1, rCAP-1, and S70D in solution were very similar.17 Therefore, the effect of the chemical structures of the peptides on CaCO3 mineralization could be examined. S70D also induced a fracture of CaCO3 crystals, and rounded crystals were produced at a concentration of 3.0 × 10-3 wt% (Figure 3). The surface morphology of the crystal was closer to that formed by natural CAP-1 than that
4064 Crystal Growth & Design, Vol. 8, No. 11, 2008
Figure 3. SEM images of CaCO3 crystals grown on the glass substrates in the presence of S70D: (a) 0.3 × 10-3 wt%; (b) 1.0 × 10-3 wt%; (c) 3.0 × 10-3 wt%, ∆N: (d) 0.3 × 10-3 wt%; (e) 1.0 × 10-3 wt%; (f) 3.0 × 10-3 wt%, and ∆C: (g) 0.3 × 10-3 wt%; (h) 1.0 × 10-3 wt%; (i) 3.0 × 10-3 wt%.
with rCAP-1. To specify the acidic part of CAP-1 that is effective for crystallization, we conducted CaCO3 crystallization on glass in the presence of recombinant peptides named ∆N and ∆C. The peptides, ∆N and ∆C lack the 17 N- and C-terminal residues of rCAP-1, respectively. In the primary structure of CAP-1, the acidic residues are separately distributed in the N-terminal region, whereas they repeat in the C-terminal region. Both peptides have the chitin binding region and the same number of acidic residues. For ∆N and ∆C, these peptides are not effective in the inhibition of crystallization and only rhombohedral crystals with fractured edges were formed even at a concentration of 3.0 × 10-3 wt%. These results indicate that the acidic part, especially the 70th residue of the peptide, plays an important role in crystallization. CaCO3 Crystallization on Chitin Matrices. In our previous study,13 the uniaxially oriented CaCO3 crystals composed of granular nanocrystals were formed in the presence of CAP-1, whereas rCAP-1 induced uniaxially oriented CaCO3 crystals composed of block-shaped calcite crystals about 200 nm in size on chitin matrices. We have further studied the cooperative effects of the peptides and chitin on CaCO3 crystallization, on the chitin matrices in the presence of S70D, ∆N and ∆C (Figure 4). Figure 4a,b shows SEM images of CaCO3 crystals grown on the chitin matrix in the presence of S70D. S70D also induced oriented calcite crystals on chitin matrices, as was observed for CAP-1 and rCAP-1. The surface of the crystalline CaCO3 assembles formed in the presence of S70D was composed of small granular crystals. The effect of S70D was similar to natural CAP-1 than rCAP-1. The fact that substitution of neutral serine by aspartic acid leads to the formation of CaCO3 crystals that are smaller than those obtained in the presence of rCAP-1 suggested that the 70th residue was active, interacting site of the peptide with calcium ions or ACC surfaces. CaCO3 crystals grown on the chitin matrices in the presence of ∆N and ∆C are shown in Figure 4c,d and Figure 4e,f, respectively. The crystals formed in the presence of ∆N
Yamamoto et al.
Figure 4. SEM images of CaCO3 crystals grown on the chitin matrices in the presence of (a, b) S70D, (c, d) ∆N, (e, f) ∆C (3.0 × 10-3 wt%). (b, d, f) are magnified images of (a, c, e), respectively.
were uniaxially oriented and composed of block-shaped crystals with a diameter of approximately 300 nm. The effects of ∆N on CaCO3 mineralization were very similar to those of rCAP-1, although ∆N lacked 17 residues of rCAP-1. In contrast, the calcite crystal formed in the presence of ∆C was a fractured rhombohedral structure. It seems that the effects of ∆C on the inhibition of crystallization were weaker than those of ∆N. These results suggest that the peptides interact with Ca2+ ions at the C-terminal part in which repetitive acidic residues exist. The N-terminal part has little effect on crystallization, although it contains as many acidic residues as the C-terminal region. It is interesting that ∆N was as active as rCAP-1 on chitin matrices despite its inactivity on glass substrates. Inoue et al. showed that the CD spectrum of ∆N in dilute solution was different from those of other peptides, suggesting that the N-terminal part contributes to maintaining the peptide conformation. ∆N may be activated by the conformational change due to the binding to chitin. The dose-dependent effects of the peptides were also examined (see Supporting Information). Conclusion We have shown that the effects of the molecular structures of an acidic peptide obtained from the exoskeleton of the crayfish and its derivatives on CaCO3 crystallization. For these peptides, the C-terminal acidic region had greater effects on CaCO3 crystallization than did the N-terminal acidic region. The 70th phosphoserine residue also had effects on the mineralization. These results show that not all of the acidic parts greatly affect CaCO3 crystallization. A study on the relationship between individual acidic motifs and their function is essential for further understanding of the proteins involved in biomineralization. This approach may be useful for the development of highly functional hybrid materials. Acknowledgment. This study was partially supported by Grant-in-Aid for the Global COE Program for Chemistry
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Innovation (T.K. and Y.Y.), Exploratory Research (No. 20655022) (T.K.), and Encouragement of Young Scientists B (No. 19750106) (T.N.) from the Ministry of Education, Culture, Sports, Science and Technology. Partial financial support by Grant-in-Aid for Creative Scientific Research (No. 17GS0311) (H.N.) from the Japan Society for Promotion of Science is also acknowledged. We are grateful to Dr. Yuya Oaki for helpful discussions. Supporting Information Available: (Figure S1) SEM images of CaCO3 crystals grown on the chitin matrices in the presence of S70D, ∆N and ∆C (1.0 × 10-3 wt%) and without any additives. This material is available free of charge via the Internet at http://pubs.acs.org. (9)
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