Motif-Programmed Artificial Extracellular Matrix - American Chemical

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Motif-Programmed Artificial Extracellular Matrix Katsutoshi Kokubun,†,‡ Kenji Kashiwagi,‡,§ Masao Yoshinari,⊥ Takashi Inoue,† and Kiyotaka Shiba*,‡,§ Department of Clinical Pathophysiology, Division of Oral Implants Research and Oral Health Science Center, Tokyo Dental College, 1-2-2, Masago, Mihama-ku, Chiba, 261-8501 Japan, Division of Protein Engineering, Cancer Institute, Japanese Foundation for Cancer Research, Koto, Tokyo 135-8550, Japan, and CREST, Japanese Science and Technology Agency, c/o Cancer Institute, Tokyo, Japan Received June 12, 2008; Revised Manuscript Received July 18, 2008

Motif-programming is a method for creating artificial proteins by combining functional peptide motifs in a combinatorial manner. This method is particularly well suited for developing liaison molecules that interface between cells and inorganic materials. Here we describe our creation of artificial proteins through the programming of two motifs, a natural cell attachment motif (RGD) and an artificial Ti-binding motif (minTBP-1). The created proteins were found to reversibly bind Ti and to bind MC3T3-E1 osteoblast-like cells. Moreover, although the interaction with Ti was not covalent, the proteins recapitulated several functions of fibronectin, and thus, could serve as an artificial ECM on Ti materials. Because this motif-programming system could be easily extended to create artificial proteins having other biological functions and material specificities, it should be highly useful for application to tissue engineering and regenerative medicine.

Introduction Interactions between cells and the extracellular matrix (ECM) are crucially involved in the processes of homeostasis, morphogenesis, and organ repair. Although the ECM primarily functions as a scaffold for cell growth, recent studies have shown that interactions between cells and ECM evoke a variety of intracellular signals that are necessary for cell maintenance and differentiation ECM.1 For example, the integrins are a family of heterodimeric (Rβ) cell surface proteins composed of different R (18 variants) and β (8 variants) subunits that define the proteins’ ligand specificities.2 Among these, eight integrins (R5β1, Rvβ3, etc.) are known to recognize the arginine-glycineaspartic acid (RGD) triplet shared in some ECM proteins (e.g., fibronectin and vitronectin).2 The binding of RGD induces the clustering of integrins along with other molecules, which organize into “focal adhesion complexes” in the cell. This in turn triggers a cascade of overlapping reactions, including cell attachment, cell spreading, cytoskeletal reorganization, and so on.3 In this way, interactions between cells and ECM intermediate between the exterior environment and such interior cellular activities as migration, survival, proliferation, and differentiation.4-8 At present, the development of artificial ECM is being urgently pursued in the fields of regenerative medicine and tissue engineering.9,10 Earlier studies have already shown that short peptides, including the RGD triplet, are able to recapitulate certain aspects of ECM when immobilized on the proper polymeric materials.11,12 Complementary studies, in which soluble RGD peptide was shown to inhibit the interaction between cells and the ECM, support the functional independence of the sequence;12 however, the full functionality of the ECM * To whom correspondence should be addressed. Fax: 81-3-3570-0461. E-mail: [email protected]. † Department of Clinical Pathophysiology and Oral Health Science Center, Tokyo Dental College. ‡ Division of Protein Engineering, Cancer Institute. § CREST, Japanese Science and Technology Agency. ⊥ Division of Oral Implants Research, Oral Health Science Center, Tokyo Dental College.

is not recapitulated by RGD alone.13 Moreover, it is known that manifestation of a functional RGD motif requires that the peptide be properly displayed on supporting materials. For instance, the NH2-RGD-COOH tripeptide shows no affinity for integrins, but elimination of the C-terminal carboxyl group or addition of extra amino acids at both ends reactivates the latent functionality of RGD.14 This capriciousness of RGD and other peptide motifs has hampered the rational design of motif-based artificial biomedical materials such as ECM.15-17 Motif-programming is a method for creating multifunctional artificial proteins by combining two or more functional motifs.16 Motifs are often short amino acid sequences within natural proteins (the aforementioned RGD peptide is one such natural motif) that are associated with particular biological functions. Motifs also can be created de novo using molecular engineering.18 In particular, peptide aptamers, which have been isolated as specific binders against various in organic targets, are believed to be promising motif blocks for creating novel biomaterials through motif-programming.19-21 It is now known, however, that simple arithmetic addition does not always work with motifprogramming, for example, simple conjugation of motif-A and -B does not always result in a bifunctional peptide-AB.15 To solve this nonlinearity in motif-programming, we have been employing a combinatorial approach, which we called MolCraft.22 In MolCraft, we prepare a library of artificial proteins that contain multiple motifs in various numbers and orders, from which clones having the desired functions are selected. One of our primary research aims is to establish a system with which we would be able to rationally design and construct artificial ECM capable of sending desired signals to cells. In our first attempt to accomplish this purpose, we used MolCraft to construct artificial proteins that endowed titanium (Ti) surfaces with the ability to bind cells (Figure 1). To immobilize biomolecules on the surface of titanium, various linking methods have been proposed.23 For instance, Schuler et al. immobilized RGD(SP) peptide on Ti using poly(-lysine)-graft-poly(ethylene glycol) (PLL-g-PEG) and showed that the coating resulted in the increased numbers of attached cells and the spreading of

10.1021/bm800638z CCC: $40.75  2008 American Chemical Society Published on Web 10/01/2008

Motif-Programmed ECM

Figure 1. Schematic representation of an artificial protein intermediating between the surface of a Ti substrate and cells. Artificial proteins are shown as yellow lines containing a Ti binding motif (minTBP-1, green lines) and a cell binding motif (RGD, red lines).

the cell’s morphology.24 Generally, the immobilization without loosing biological activities is challenging goal because hydrophobic interaction between biomolecules and Ti surface may result in the inactivation of the biomolecules. They used PLLg-PEG to avoid this problem. Our approach is to use Ti-binding peptide for the immobilization. Because this binding peptide is known to reversibly associate the Ti-surface, the interaction between Ti and biomolecules would not be expected to be too strong. In this paper, we created artificial protein by combining this Ti binding motif and RGD motif.

Materials and Methods Ti Plates. Commercially available pure wrought Ti plates (JIS, Japan Industrial Specification H4600, 99.9 mass%, Ti) were used in this study. The plates were polished using a buff and then ultrasonically cleaned in ethanol and distilled water. Before each experiment, the plates were treated for 10 min with a UV/ozone cleaner (ProCleaner, BioForce Nanosciences Inc., Iowa). Then, using a profilometer (Handy Surf 130A, Tokyo Seimitsu, Tokyo), the surface roughness (Ra) of the plates was measured to be 0.10 ( 0.01 mm. Cells. Preosteoblastic MC3T3-E1 cells were generously provided by Dr. K. Imamura (Cancer Institute, Japanese Foundation for Cancer Research), and a human keratinocyte line (HaCaT) was provided by Dr. R. Yao (Cancer Institute, Japanese Foundation for Cancer Research). The MC3T3-E1 cells were maintained and expanded in R-MEM (GIBCO-BRL/Invitrogen, Carlsbad, CA) supplemented with 10% FBS (JRH Bioscience, Lenexa), 100 µg/mL penicillin (Banyu Pharmaceutical Co., LTD, Tokyo), and 100 units/mL streptomycin (Meiji Seika Kaisha, LTD, Tokyo) in a continuous culture at 37 °C under a humidified atmosphere containing 5% CO2 and 95% air. HaCaT cells were grown in Dulbecco’s modified Eagle’s medium (DMEM; GIBCO-BRL) supplemented with 10% FBS, 100 µg/mL penicillin, and 100 units/ mL streptomycin at 37 °C and 5% CO2. The cells were subcultured every 3-4 days. Design and Construction of Artificial Proteins. In this study, we wished to create a set of artificial proteins that would attach to the surface of Ti plates, thereby endowing them with a capacity for cell attachment. Ti is already being widely used in artificial joints and dental implants.25 In fabric form, moreover, Ti is expected to serve as sturdy scaffold for tissue regeneration area.26 In that context, we decided to use our motif-programming approach to biologically functionalize the surface of Ti. To endow Ti with the capacity for cell attachment, an artificial protein must function in two ways: (i) it must specifically bind to the surface of Ti and (ii) it must adhere to the surface of cells (Figure 1). For this purpose, we focused on two motifs: the aforementioned RGD cell attachment motif27 and minTBP-1, a hexapeptide (RKLPDA) that corresponds to the core region of the 12-amino acid peptide TBP-1, which was artificially created as a Ti-binding peptide using a peptide phage system.19 minTBP-1 appears to bind electrostatically to the oxidized surface of Ti via its R1 and D5 residues, and its ability to bind Ti can be transferred to other proteins. For example, ferritin, a hollow spherical protein composed of 24 subunits, was endowed with Ti binding activity by fusing minTBP-1 to the N-terminal end of the

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subunit.28 Thus, the ability to bind Ti can be programmed into foreign molecules using the minTBP-1 motif. In the present study, therefore, we designed a 36-bp microgene, MG-66, so that it encoded minTBP-1 and RGD in its first and third reading frames, respectively. Using the CyberGene program,22 codons for this microgene were chosen so that peptides encoded by the different reading frames included few charged amino acids. Starting with this microgene as a building block, we synthesized two MPR (microgene polymerization reaction) primers (KY-1528: 5′- GGTCGGGGGGACAGCTTCGCAAACA-3′; KY-1529: 5′- AAGCGTCCGGGAGTTTGCGAAGCTGA-3′) and prepared a library of artificial proteins by tandemly polymerizing the microgene using the previously described MPR method.29 Because the MPR conditions allowed for random insertion or deletion mutations at the junctions of the microgene units, the translational products of the obtained microgene-polymers were combinatorial polymers of three peptides encoded by the single microgene. The library thus encoded a variety of proteins that contained different numbers of the minTBP-1 and RGD motifs in various orders. We initially cloned 14 arbitrarily chosen polymers into expression vectors, but ultimately focused on three proteins, #KB089, #KB094, and #KB103, because they were highly expressed in Escherichia coli and were soluble under physiological conditions. The proteins were purified under denaturing conditions, as previously described,30 after which they were stored in 50 mM Tris-acetate buffer (pH 4.0) containing 100 mM NaCl and 1 mM EDTA. We determined the molecular weights of the proteins using a MALDI-TOF AXIMA (Shimadzu, Kyoto) and confirmed that the calculated values agreed well with those observed. Quartz Crystal Microbalance (QCM) Measurements. For analyses of the binding of artificial proteins to a Ti surface, we used a QCM-D instrument (QCM-D300, Q-Sense AB, Go¨tenborg) and a Ti sputter-coated QCM sensor. Prior to making measurements, the sensor was cleaned for 10 min using a UV/ozone surface treatment system. The measurements were carried out at 25 °C, and the data for analysis were collected at 14.8 MHz. The sensor was first equilibrated with TBS-T [TBS (50 mM Tris-HCl, pH 7.5, 150 mM NaCl) containing 0.5% Tween 20 (Sigma, St. Louis)], after which solution containing 0.1 mg/mL of protein in TBS-T was injected. The data were modeled using Q-tools software. Cell Adhesion Assays. Ti plates (6 × 6 × 1 mm) in 48-well culture plates (Falcon, Franklin Lakes) were incubated for 1 h at 37 °C in 200 µL of TBS containing one of the artificial proteins. The plates were then blocked for 1 h at the same temperature with phosphate-buffered saline (PBS; GIBCO-BRL) containing 1% bovine serum albumin (BSA; Iwai, Tokyo,). MC3T3-E1 cells were collected by trypsinization and then suspended in serum-free R-MEM at a density 2 × 105 cells/mL. Thereafter, 500 µL aliquots of the cell suspension were seeded onto the Ti plates and incubated for 1 h at 37 °C in a 5% CO2 incubator. After washing three times with PBS, cells that had attached to the plates were fixed and stained for 30 min with 0.4% crystal violet (Wako, Osaka) in methanol (w/v). The plates were then washed with distilled water, the stained cells were solubilized with 1% SDS in methanol, and the absorbance of each well was measured at 595 nm using a model 680XR microplate reader (Bio-Rad, Hercules). Immunofluorescence Microscopy. Focal adhesions and Actin stress fibers in the adherent MC3T3-E1 cells were visualized using standard fluorescence microscopy. Cells were seeded onto Ti plates (10 × 10 × 1 mm) to a density of 1 × 105 cells/well in 24-well tissue culture plates (Iwaki, Tokyo) and incubated for 1 h in serum-free medium. Thereafter, the cells were rinsed with PBS, fixed in 4% paraformaldehyde (Nacalai, Kyoto) for 20 min at room temperature, permeabilized for 10 min in PBS containing 0.1% Triton X-100 solution, and then washed twice with PBS. Nonspecific binding was blocked with PBS containing 1% BSA. An FITC-conjugated antipaxillin antibody (1:200 dilution; BD Biosciences, San Jose) was used to localize the punctate focal adhesion structures at the cell-ECM interface. Filamentous Actin (F-Actin) stress fibers were visualized using rhodamine-conjugated phalloidine (1:40 dilution; Molecular Probes, Carlsbad).

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Immunoblot Analysis. MC3T3-E1 cells were seeded onto proteincoated Ti-plates to a density of 2 × 105 cells/well in 24-well dishes. After incubation for 1 h, the cells were washed twice in PBS and then lysed for 5 min with RIPA buffer (Sigma). The samples were then scraped from the plates, and the total protein concentration in the collected samples was assayed using a Pierce BCA protein assay kit. Western blots were obtained by transferring the proteins to a polyvinylidene difluoride (PVDF) membrane for 7 min using an iBlot (Invitrogen). The membrane was then blocked for 30 min at room temperature in Blocking One-P (Nacalai), after which it was incubated overnight at 4 °C with a primary antibody against either active FAK [mouse anti-FAK (phosphorylated Y397, Tyr397) IgG; BD Transduction Laboratories, San Jose, CA] or total FAK [mouse anti-FAK mAb IgG (clone 77); BD Transduction Laboratories]. The blots were then washed three times for 5 min each with TBST buffer [10 mM Tris (pH 7.5), 0.1 M NaCl, 0.1% Tween 20] and incubated with horseradish peroxidase (HRP)-conjugated goat antimouse IgG (1:2000 dilution; BIO-RAD). The bands were developed by adding an enhanced chemiluminescent substrate (ECL Plus, Amersham-Pharmacia, Arlington Heights, IL). Alkaline Phosphatase (ALP) Staining. To assess the osteoblastic differentiation of the MC3T3-E1 cells, the cells were seeded onto uncoated and protein-coated Ti plates to a density of 1 × 105 cells/ well in 24-well plates and incubated for 4 days in differentiation medium [R-MEM culture medium containing 10% FBS supplemented with 10 mM β-glycerophosphate (Sigma), 50 µM ascorbic acid (Sigma), and 100 nM dexamethasone (Sigma)]. The cells were then stained for ALP using BCIP/NBT (Sigma) according to the manufacturer’s instructions. Cell Spreading Assay. HaCaT cells were grown on Ti plates coated with artificial proteins in DMEM for 2 h at 37 °C. To visualize the organization of cytoskeleton, the F-Actin was stained with rhodamineconjugated Phalloidine. The major axes of cells were estimated using Image-J software (National Institutes of Health, Bethesda, MD).

Results Design and Synthesis of Artificial Proteins. We initially designed a microgene, MG-66, that encoded minTBP-1 (RKLPDA) and RGD in its first and third reading frames, respectively (Figure 2a). Because minTBP-1 interacts with Ti via an electrostatic interaction, we chose codons for the microgene so that the number of charged amino acids within the encoded peptides would be limited. Based on the DNA sequence of the designer microgene, two oligonucleotides with overlapping regions and a mismatched base pair at their 3′ ends were designed and synthesized as MPR primers (Figure 2b). When these primers are used, polymers of the microgene were prepared under the previously described MPR conditions,29 which allowed random insertion and deletion mutations at junctions of the microgene (Figure 2b). Because of these mutations, the resultant polymers made up a library composed of combinatorial polymers of three peptides encoded by MG-66. Because minTBP-1 and RGD were embedded in the different reading frames of the microgene, the library contained artificial proteins that carried different numbers of these motifs in various orders. The artificial genes were cloned into an E. coli plasmid vector, after which the expression level and solubility of the purified expressed proteins were tested (a total of 14 were tested). Based on the results of this initial screening, we selected three clones, pKB089, pKB094, and pKB103 that expressed large quantities of translated product (#KB089, #KB094, and #KB103, respectively) with good solubility for further characterization (Figure 2c). The molecular weights (MWs) and isoelectric points (pIs) of the proteins, and the number of motifs present, are summarized in Table 1. The purified proteins were monodispersed at a concentration of 1 mg/mL in a DLS assay (Supporting

Figure 2. Motif-programmed microgene and construction of artificial proteins. (a) The designer microgene used in this study (MG-66), with the RGD and minTBP-1 motifs shown in red and green boxes, respectively. The three reading frames are colored green, gray, and red. (b) Scheme of the construction of artificial proteins. Two MPR primers were designed from MG-66; they yielded polymers of MG66 (designated as MG in the figure) upon performance of the MPR, which allowed random insertion and deletion mutations at junctions of the microgene. The resultant polymers made up a library composed of combinatorial polymers of three peptides encoded by MG-66. (c) The three clones focused on in this study.

Information, Figure S1a), and they gave CD spectra typical of disordered structures (Supporting Information, Figure S1b), which are often observed with artificial proteins created using our method.16,30,31 Binding of Artificial Proteins to Ti Surfaces. We next investigated the interaction between the purified artificial proteins and a Ti surface using a QCM equipped with a Ti sensor. Upon the injection of 0.1 mg/mL #KB089, #KB094, or #KB103 into the measurement cell, an immediate reduction in the resonance frequency of the sensor was observed (Figure 3). Because the energy dissipation shifts with the injections were less than 1 × 10-6, Sauerbrey’s equation32 could be applied, and the calculated mass gains were 136, 102, and 83 ng/cm2 for #KB089, #KB094, and #KB103, respectively (note that the calculated mass includes waters bound to the proteins). In this experiment, we also used lysozyme, fibronectin, and an artificial



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Table 1. Properties of the Artificial Proteins Used in this Study amino acid composition a

b

c

motif composition d

name

total residues

MW

pI

positively charged

negatively charged

RGD

TBP-1

#KB089 #KB094 #KB103 #JZ018 fibronectin

132 192 113 142 2386

13867 19824 12068 16607 262606

11.75 12.30 12.06 12.36 5.45

17 26 16 31 202

7 7 6 6 259

3 5 4 0 1

3 2 1 5 0

a MW: calculated from nucleotide sequences. For #KB089, #KB094, and #KB103, the molecular weights were determined to be 13867, 19824, and 12088, respectively, by MS (Ciphergen). b pI: calculated from http://tw.esxpasy.org/tools/protparam.html. c Positively charged: Lys and Arg. d Negatively charged: Asp and Glu.

Figure 3. QCM analyses of the interaction between Ti and the artificial proteins. Injection of a protein (0.1 mg/mL; red, #KB089; blue, #KB094; green, #KB103; gray, #JZ018; and black, lysozyme) into the QCM measuring chamber (black arrowhead) immediately reduced the resonance frequency of the sensor (y-axis), which indicated the association of the protein with the Ti surface. Dissociations were observed as increases of resonance frequencies upon injections of TBS-T into the chambers (gray arrowheads).

protein, #JZ018, as controls. Lysozyme is a globular hydrophilic protein that is positively charged under the conditions used for the artificial proteins. Fibronectin is a representative ECM protein and has one RGD motif in its sequence. #JZ018 is an artificial protein that was programmed with minTBP-1 but did not contain an RGD motif (Supporting Information, Figure S1c). Injection of lysozyme reduced the resonance frequency by only 2.5 Hz, indicating a weak association with the Ti sensor. Both #JZ018 and fibronectin reduced the frequencies, and the former induced increase in mass of 100 ng/cm2. The association between fibronectin and the Ti sensor brought an energy dissipation shift of >3.5 × 10-6, which deviated from Sauerbrey’s model and prevented us from evaluating the molecular gain of the sensor (Supporting Information, Figure S2a). Increases in resonance frequency were observed upon injection of buffer into the protein-associated Ti sensor (Figure 3), indicating that the interactions between the minTBP-1programmed proteins and the Ti sensor were reversible. By contrast, the QCM analysis showed that there was only limited dissociation of fibronectin from the Ti sensor, suggesting its interaction was largely irreversible (Supporting Information, Figure S2a). The structures of the artificial proteins created with MolCraft are inherently repetitive, which hinders mutational analyses using site-directed mutagenesis. In addition, the microgene that has mutated minTBP-1 or RGD must introduce different amino acid sequences in other reading frames. Consequently, we could not use appropriate mutants to directly prove the involvement of the embedded minTBP-1 motif in the binding of the proteins to the Ti. Nonetheless, we suggest the embedded minTBP-1

motif was responsible for the interaction between the proteins and Ti because (i) the rapid association of the proteins with the Ti surface and relatively slow dissociation are consistent with the properties observed in minTBP-1-ornamented ferritin molecules,28 and (ii) only one-third as much artificial protein (by weight) was adsorbed onto a gold surface (Supporting Information, Figure S2b). Sano et al. previously reported that the ability to bind Ti can be transferred to a foreign molecule (ferritin) by appending minTBP-1 to the ends of the molecule’s subunits.28 The present data further demonstrate that Ti-binding ability also can be transferred to a protein by embedding the motif in the middle of the protein’s sequence. Cell Attachment Activities of Artificial Proteins. To characterize the proteins’ ability to mediate cell attachment, we seeded MC3T3-E1 cells, a mouse osteoblast-like cell line known to express R5β1 and Rvβ3 integrin,33 onto protein-coated Ti plates. To make comparisons, some plates were coated with #JZ018, which does not contain the RGD motif or fibronectin. Using Western blotting to monitor the presence of the artificial proteins on the Ti plates (Supporting Information, Figure S3), we observed that, in PBS, the proteins remained on the plates for at least two days, and that fibronectin detached more slowly than the artificial proteins (Supporting Information, Figure S3). These qualitative analyses of detachment rates were compatible with the results of the QCM analyses (Figure 3 and Supporting Information, Figure S2). Although quantification of the protein coating of the Ti plates was not possible, we were able to treat Ti plates with increasing concentrations of protein and then count the numbers of MC3T3E1 cells attached to these plates after 1 h for incubation. As shown in Figure 4, all of the proteins tested mediated cell attachment to the Ti in a concentration-dependent manner. The numbers of cells attached to the #KB089- and #KB103-coated Ti plates were equivalent to those attached to fibronectin-coated plates and twice that attached to #JZ018-coated plates. In addition, #KB094, which contained five copies of the RGD motif, showed an even greater capacity for cell attachment than fibronectin. These artificial proteins thus appear to have a capacity for cell attachment that is compatible to, or even better than, that of fibronectin. Artificial Proteins Induce Formation of Stress Fibers and Focal Contacts. The cell attachment experiment summarized in Figure 4 enabled us to evaluate only the initial phase of the interaction between cells and matrix; consequently, the results do not necessarily indicate that the functionality of the artificial proteins is equivalent to that of fibronectin. The interaction between fibronectin and integrins is followed by the formation of F-Actin stress fibers and focal adhesion complexes.34,35 To test whether the artificial proteins can induce this second phase of cellular events, we used immunohistochemistry and fluorescence microscopy to assess the formation of Actin fibers and focal adhesions in the cells. As shown in

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Figure 6. Western blotting of extracts from cells grown on proteincoated Ti plates. Antibodies against phosphorylated FAK (Tyr397, P-FAK) and total FAK (T-FAK) were used. Ti indicates an uncoated Ti plate. Relative strengths of signals estimated by Image-J were shown.

Figure 4. Adhesion of MC3T3-E1 cells to Ti plates coated with the indicated proteins. Ti plates were coated with the indicated concentrations of #KB089, #KB094, #KB103, #JZ018, or fibronectin and then incubated with MC3T3-E1 cells for 1 h at 37 °C. The relative numbers of cells attached to the Ti plates were quantified by measuring the absorbance at 595 nm (y-axis) after staining the cells with crystal violet. Symbols and bars represent the means ( SD of three independent experiments: red, #KB089; blue, #KB094; green, #KB103; gray, #JZ018; orange, fibronectin (FN).

Figure 5. Cytoskeletal reorganization and focal contact formation in cells grown on Ti plates coated with an artificial protein. MC3T3-E1 cells were allowed to grow for 1 h on Ti plates coated with the indicated artificial proteins or fibronectin (FN) in DMEM. Cells were then stained with rhodamine-phalloidine to detect F-Actin (left, red) or antipaxillin antibody to detect focal adhesions (right, green). The scale bar represents 20 µm.

Figure 5, cells grown on fibronectin-coated plates contained a well-organized network of F-Actin stress fibers and formed focal adhesions that were identified by staining with an antipaxillin antibody. Similar Actin fiber networks and adhesion foci were observed in cells grown on #KB094- or #KB103-coated plates, indicating that these proteins acted as ECM to the same degree

as fibronectin. By contrast, formation of Actin fibers and adhesion foci was disrupted in cells grown on bare metal plates or plates coated with #KB089 or #JZ018. FAK and Phosphorylated FAK Expression on Artificial Proteins. The formation of the focal adhesions and Actin fibrils in cells grown on #KB094- or #KB103-coated Ti plates suggested the interaction of integrins and the artificial proteins triggered the appropriate intracellular signaling. To confirm this idea, we evaluated the phosphorylation state of FAK, which is known to be phosphorylated within focal adhesion complexes and to play a central role in the signal transduction there.35 We examined the phosphorylation patterns of FAK in the cells grown on the artificial ECM using antibodies that recognized the phosphorylated tyrosine (Tyr397) in FAK. We found that the level of Tyr397 phosphorylation was approximately three times higher in cells grown on #KB094 than on uncoated plates and those coated with #JZ018 (Figure 6). Induction of Osteoblastic Differentiation by Artificial Proteins. We have shown that artificial proteins created from “cell attachment” and “Ti-binding” motifs induce some of the same cellular responses as natural fibronectin. This is despite the fact that the structural features of the artificial proteins and fibronectin markedly differ (the former have a disordered structure (Figure 2), while the latter is composed of β-structure36), as does the mode of interaction with Ti (minTBP-1 binds electrostatically to Ti, while fibronectin must associate with Ti through a hydrophobic interaction37). Could these physicochemical properties differentiate the biological functions of these proteins? To address this question, we compared the effects of artificial proteins and fibronectin on higher cellular phenotypes. The MC3T3-E1 line are osteoblast-like cells that can be induced to differentiate by ascorbic acid and β-glycerophosphate and dexamethasone.38 A key marker of this differentiation is the induction of ALP expression. When we incubated MC3T3-E1 cells on protein-coated plates for 4 days, we found that high levels of ALP activity were expressed in cells grown on the #KB094- and fibronectin-coated Ti plates, but only moderate activities were seen in cells grown on uncoated plates or those coated with #KB089, #KB103, or #JZ018 (Figure 7). The elevated phosphorylation in FAK-Tyr397 grown on the #KB094coated Ti plates (Figure 6) was apparently correlated with this higher ALP induction. Thus, #KB094 and #KB103, which were identical with respect to there abilities to stimulate formation of adhesion foci (Figure 5), differ in there ability to stimulate higher biological activity (Figures 6 and 7). Spreading of HaCaT Keratinocytes on Artificial Proteins. To determine whether any of the artificial proteins possess functions not possessed by fibronectin, we next investigated their effects on the growth of HaCaT epithelial cells, which have been shown to express integrins that specifically bind to the



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Figure 7. ALP expression in MC3T3-E1 cells grown on Ti plates coated with artificial proteins. Cells were plated on protein-coated Ti plates and incubated for 4 days under conditions that induce ALP expression. Relative expression levels were then estimated by staining for ALP. The scale bar represents 100 µm.

RGD motif.39 We grew HaCaT cells on protein-coated Ti plates and estimated “cell-spreading” by measuring the planar areas of the cells (Figure 8a). Interestingly, cells grown on the #KB094- and #KB103-coated plates were significantly larger in area than those grown on the fibronectin-coated plates (Figure 8b). In addition, after incubation for 7 days, greater numbers of cells were present on the Ti plates coated with #KB094 than on those coated with fibronectin (Supporting Information, Figure S4). Finally, we used time-lapse microscopy to monitor the movement of cells on protein-coated plates. Interestingly, cells on fibronectin moved around on the plate before settling down to the substrate, whereas cells on artificial proteins showed no such active movements and adhered the substrate earlier than cells on fibronectin (Supporting Information, movie S1). Taken together, these findings suggest that artificial proteins may more effectively mediate some cellular responses than the natural proteins.

Discussion The aim of this study was to confirm the utility of “motifprogrammed”40 artificial proteins for artificial ECM development. Recent advances in genome science have revealed a variety of naturally occurring motifs or short peptide sequences associated with specific biological functions. In addition, evolutionary molecular engineering has enabled us to create artificial motifs that interact with inorganic materials, a good example of which is minTBP-119 used in this study. By combining these natural and artificial motifs, we are now able to create liaison proteins able to endow inorganic materials with protein-based biological activities, which could eventually lead to the development of novel composite materials having multiple functions and supporting high levels of performance. In the present study, we focused on two motifs: RGD,27 a naturally occurring cell attachment motif, and minTBP-1,19 an artificial Ti-binding motif. By combining these two, we were able to create artificial proteins that endowed Ti surfaces with a capacity for cell attachment. Ti has been widely used as an implant material25 because of its high resistance to corrosion,

Figure 8. Effects of artificial proteins on cell spreading. (a) HaCaT cells grown on Ti plates coated with the indicated artificial protein or fibronectin (FN) were stained with rhodamine-phalloidine to visualize F-Actin. The scale bar represents 20 µm. (b) The areas of the spreading cells seen in (a) were quantified using image analysis software. Bars depict the means ( SD form four samples.

its biocompatibility, and its immunologic inertness. Controlled attachment of cells to Ti is crucial for the development of the next generation of Ti-based materials. Consequently, the establishment of a method with which to stimulate cell attachment to Ti surfaces is urgently required. It has been argued that this can occur without modification as long as the system contains serum, because serum contains fibronectin, vitronectin, and various cytokines that eventually help cell attachment.41 From a clinical viewpoint, however, it would be desirable to have the ability to rationally endow the surface of materials with selected biological activities, irrespective of the presence of serum. We first assessed the functionality of our three artificial proteins by comparing their abilities to mediate cell attachment with the ability of fibronectin and an artificial protein lacking the RGD motif. The results showed that all three programmed

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proteins mediated cell attachment as well as, or even better than, fibronectin (Figure 4). Moreover, the observed adhesive interactions between cells and the artificial proteins were mediated by integrins on the cell membrane, which interacted with RGD sequences in the artificial proteins. This in turn stimulated the formation of well-developed F-Actin stress fibers and focal adhesions in the cells grown on #KB094 or #KB103 (Figure 5). This response along with the observed upregulation in FAK phosphorylation (Figure 6) strongly supports the notion that a biological interaction occurred between these two proteins and the cells. Consistent with that idea, #KB094, like fibronectin, even supported osteoblastic differentiation of MC3T3-E1 cells on protein-coated Ti plates (Figure 7). Although the artificial proteins created for this study and fibronectin all contain the RGD motif, in other ways they differ remarkably. Whereas the artificial proteins are small, soluble, structurally disordered, and have a high pI, fibronectin is large, easy to precipitate, rich in secondary structure, and has a low pI (Table 1).36 Furthermore, their modes of interaction with Ti differ. The three artificial proteins rapidly associated with Ti surfaces and then slowly dissociated from them. This behavior most likely reflects the inherent properties of the embedded minTBP-1 motif,28 which reversibly binds Ti through an electrostatic interaction.19,42 By contrast, fibronectin slowly bound to Ti and, in QCM analyses, most of bound protein remained attached after washing the sensor (Supporting Information, Figure S2a), which suggests hydrophobic interactions dominate the binding of fibronectin to Ti surfaces. One of our interests in this study was whether or not proteins only weakly and reversibly bound to Ti could mediate integrin signaling in attaching cells. Our data indicate that the artificial proteins recapitulated all of the functions of fibronectin tested so far, which included cell adhesion (Figure 4), cytoskeletal reorganization (Figure 5 left), the formation of focal adhesions (Figure 5 right), phosphorylation of FAK (Figure 6), and the induction of ALP expression (Figure 7). Although additional studies will be required to determine the biological importance of these observations, we believe the reversible immobilization method is well suited for the development of elaborate new biomaterials because most biological activities proceed in a reversible manner. Notably, some of our artificial proteins showed a greater ability to mediate ECM-induced intracellular signaling than fibronectin and would therefore seem worthy of further attention. For example, HaCaT cells grown on #KB094 and #KB103 were well extended, and their planar dimensions were even larger than those of cells grown on fibronectin (Figure 8). In addition, long-term incubations showed that cell growth and proliferation on these proteins was better than on fibronectin (Figure S4). Finally, cell movements on the coated substrates markedly differed on fibronectin and artificial proteins (Supporting Information, movie S1). We previously showed that a combinatorial approach brings latent motifs into full action, which highlights the fact that manifestation of a peptide motif’s full functionality is strongly influenced by its context within the artificial protein.15,17,40 This aspect of combinatorial approach differs from the rational design of molecules and, as far as our knowledge on the relationships between structure and function of polypeptides is limited, the “selection from library” method should play an important role in seeking the unexploited functions of motifs. Our current experiments were no exception. The three created proteins showed phenotypes that differed in ways that are difficult to explain simply from the number of embedded motifs (Figures

Kokubun et al.

5-8, Table 1). Most likely peptide sequences other than the embedded motifs also influence the overall activities of the proteins. For instance, our observation that the amount of #KB089 adsorbed onto a Ti surface was greatly increased when hydrophobic surfaces were not masked by Tween 20 (Supporting Information, Figure S2c) suggests some sequences of #KB089 contribute to a hydrophobic interaction with Ti and confirms the importance of the combinatorial approach to motif-programming. In this study, we embedded two motifs in a single microgene. However, as we have shown, MolCraft can scramble three40 or more motifs16 to make multifunctional artificial proteins. Because “PHSRN” motif, which has been known to bind the integrin subunit and act synergistically on the action of RGD sequence,43,44 the addition of this motif or other similar motif(s) as well as the usage of aptamer motifs against various inorganic materials could further improve and elaborate the functions of artificial ECMs.

Conclusion The immediate goal of our study was to establish a novel system that enables us to rationally endow the Ti surfaces with selected biological functions. As a proof of concept, we successfully created artificial proteins containing embedding cell attachment and Ti-binding motifs that endowed Ti with cell attachment activity. This method could be easily extended to the creation of proteins having other biological functions, such as receptor binding or antibacterial activity, just to name two, and also could be applied to the development of artificial proteins that bind to inorganic materials other than Ti. This system should thus be highly useful for application to tissue engineering and regeneration medicine. Acknowledgment. We thank Dr. K. Sano for his valuable discussion and Ms. Minamisawa for her assistance. Supporting Information Available. Methods, the results of CD and DLS analyses of the artificial proteins, QCM data for the interaction between Ti and fibronectin, immunostaining of the artificial proteins and fibronectin on Ti plates, and a timelapse movie showing cell growth on protein-coated Ti surfaces. This material is available free of charge via the Internet at http:// pubs.acs.org.

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