Construction of a bFGF-Tethered Extracellular Matrix Using a Coiled

Aug 6, 2011 - A novel method for construction of biomaterials for tissue engineering was developed. Noncovalent associations between extracellular mat...
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Construction of a bFGF-Tethered Extracellular Matrix Using a Coiled-Coil Helical Interaction Eiry Kobatake,* Ryota Takahashi, and Masayasu Mie Department of Biological Information, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology B-28 4259, Nagatsuta, Midori-ku, Yokohama, 226-8501, Japan

bS Supporting Information ABSTRACT: A novel method for construction of biomaterials for tissue engineering was developed. Noncovalent associations between extracellular matrix (ECM) and growth factors were achieved by engineering recombinant versions of both proteins that included helical peptides that could form a coiled-coil structure. The helix A peptide, which is capable of forming a coiled-coil helical structure, was fused with a matrix protein that contains a cell-adhesive RGD sequence. The helix B peptide, which is also capable of forming a coiled-coil helical structure, was fused with basic fibroblast growth factor (bFGF). Each protein retained its original activity of promoting cell adhesion and cell proliferation, respectively. These recombinant proteins associated noncovalently through coiled-coil helix formation between helix A and helix B. The resulting complex combined the functions of both proteins, and this method of joining proteins with different functionalities could be used to develop biomaterials for tissue engineering.

’ INTRODUCTION Scaffolds with signaling molecules where cells can adhere, proliferate, and differentiate efficiently are essential for tissue engineering. Biomaterials providing such an environment for cells play an important role in most tissue engineering applications. In designing materials to allow specific cellular responses, recent strategies for tissue engineering applications have focused on the design of biomimetic materials. These biomaterials, which mimic functions of the extracellular matrix (ECM), are able to interact with surrounding tissues through specific molecular recognition motifs.1,2 The incorporation of soluble signaling molecules such as growth factors and cell-binding properties into scaffold materials is one strategy to achieve biomolecular recognition of materials by cells.36 In designing these strategies, supplementary steps for chemical or physical modification of the biomaterials are required. To avoid such troublesome processes, we previously constructed a fusion protein by genetic engineering, designated ERE-EGF, which is easy to immobilize onto hydrophobic surfaces and which also enhances cell adhesion and cell proliferation.7,8 Most signaling molecules are recognized and bound to their receptors on cell surfaces. After transducing signals into cells, they are internalized by endocytosis and digested. When the signaling molecule is immobilized on a scaffold by covalent bonding, some problems such as decreased recognition efficiency by the receptor, loss of activity, or malignant transformation caused by continuous signaling should be considered. One approach to overcome these problems is to utilize noncovalent bonding between the signaling molecule and the scaffold. Some experimental approaches include tethering of a growth factor to r 2011 American Chemical Society

its substrate by engineering noncovalent bonding interactions between them. Boucher et al. utilized a coiled-coil interaction of E and K coils to immobilize epidermal growth factor (EGF) on a silicone surface.9 A polyhistidine tag has been fused with EGF in order to immobilize EGF on a Ni2+-bearing substrate.10 Some growth factors have domains that bind to the ECM. Utilizing a similar approach, the collagen-binding domain from fibronectin has been introduced into growth factor proteins.1113 These fusion proteins had higher cell-growth activity when added to collagen than did soluble forms of the same proteins when they were added to cells on a collagen matrix. This method is effective for tissue engineering, which requires collagen as an ECM; however, other binding domains are needed to apply this method to other noncollagen ECM surfaces. In order to immobilize signaling molecules to various types of ECM through noncovalent bonding interactions, the present study investigated proteins engineered to form coiled-coil dimers. Specifically, one peptide capable of forming a coiled-coil structure was fused with a growth factor protein, and another peptide with similar properties was fused with an ECM protein. A typical and well-studied coiled-coil dimer in nature is the leucine zipper of the yeast transcription factor GCN4.14 However, this protein is a homodimer, and there is no interhelical ionic interaction. Another well-studied coiled-coil structure is the heterodimer of the Fos/Jun leucine zipper.15 Unfavorable interhelical electrostatic interactions destabilize the homodimer form, thereby Received: May 12, 2011 Revised: July 24, 2011 Published: August 06, 2011 2038

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Figure 1. Design concept for the bFGF-ERE complex with a coiled-coil structure. Fusion proteins with helix A-bFGF (HA-FGF) and ERE-helix B (ERE-HB) were constructed, and they were noncovalently associated due to coiled-coil formation between the helix A and helix B moieties.

favoring heterodimer formation. On the basis of these studies, O0 Shea et al. designed two peptides, designated ACID-p1 (helix A) and BASE-p1(helix B), which associate preferentially to form a stable, parallel, coiled-coil heterodimer with a leucine zipper and with favorable electrostatic interactions.16 In this study, the ERE protein, which has been described elsewhere,7 was used as the ECM. In ERE, an RGD sequence found in the cell adhesion region was inserted between two other sequences, thus mimicking the ECM. Both of these peptide sequences contain 12 repeats of the polypeptide sequence AlaPro-Gly-Val-Gly-Val (APGVGV) designated as E. These kinds of sequence motif called elastin like polypeptides (ELPs) are found in elastin, which provides strength and flexibility in the ECM. ELPs have been paid attention as biomaterials for tissue repair.1719 On the other hand, basic fibroblast growth factor (bFGF) was used as a model signaling molecule since it has a wide variety of activities such as cell proliferation and differentiation. Helix A and helix B were fused at the N-terminus of bFGF and at the C-terminus of ERE, and the resulting proteins were designated as HA-FGF and ERE-HB, respectively. The structure of the complex formed between HA-FGF and ERE-HB through coiledcoil interactions is shown schematically in Figure 1. HA-FGF was immobilized via coiled-coil helix formation on a cell-culture plate whose surface was coated with ERE-HB, and the cell behavior on the surface was investigated.

’ EXPERIMENTAL PROCEDURES Materials. Plasmid pBluescriptSKII (-) was obtained from Toyobo. Plasmid pET32c and E. coli Bl21 (DE3) were purchased from Novagen. E. coli KRX was obtained from Promega. Synthesized DNA fragments were purchased from Texas Genomics Japan. Synthesized oligopeptides were purchased form Sigma-genosis. Restriction enzymes and ligase were purchased from Toyobo and Takara Bio. All other chemicals were of analytical grade. Construction of Plasmids. A pBS-FGF plasmid encoding a mouse bFGF gene was constructed previously. The DNA fragment encoding helix A was inserted at the 50 -end of the bFGF sequence of pBS-FGF. The helix A-bFGF fusion gene was inserted into pET32-c for expression, and the resulting plasmid was designated as pET-HA-FGF. A pBS-ERE plasmid encoding the ERE sequence was created as described elsewhere.20 This plasmid has 12 repeats of APGVGV (E12) and RGD, as well as another E12 (the ERE sequence). The DNA fragment encoding (GGGS)4 as a linker peptide was inserted at the 30 -end of the ERE sequence of pBS-ERE. The DNA fragment encoding helix B

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was inserted into the pET32c expression vector (pET32c-helix B). The fusion gene of ERE and (GGGS)4 was inserted at the 50 -end of helix B of the pET32c-helix B. The resulting plasmid was designated as pET-ERE-HB. The DNA sequences of HA-FGF and ERE-HB with their translated sequences are shown in Supporting Information (Figure S1). Protein Expression and Purification. E. coli KRX cells were transformed with plasmids and then were grown at 37 °C in an LB medium supplemented with ampicillin, to an OD660 = 0.6. After induction of protein expression with lamnose (0.1%) and isopropylthio-β-D-galactoside (1 mM), cells were cultured at 30 °C for another 4 h. Cells were harvested by centrifugation at 4 °C and then washed with PBS. After centrifugation, the cell pellet was resuspended in Bug Buster Reagent (Novagen) with benzonate nuclease (Novagen) and disrupted by gentle rotation. After centrifugation, the supernatant was applied to a TALON metal affinity resin (Clontech). After washing, the bound proteins were obtained by digesting with enterokinase (Novagen) for 16 h at room temperature. The enterokinase was removed by applying the digest onto EKapture agarose (Novagen). Cell Proliferation Activity by Soluble HelixA-bFGF Fusion Protein. To investigate the growth factor activity of helixA-bFGF (HA-FGF), human umbilical vein endothelial cells (HUVEC) were utilized. The HUVEC cell line was purchased from Kurabo and maintained at 37 °C under 5% CO2 in HuMedia EG-2 supplemented with 2% FBS, 10 ng/mL human EGF, 1 mg/mL hydrocortisone, 50 mg/mL gentamicin, 50 ng/mL amphotericin B, 5 ng/mL human bFGF, and 10 mg/mL heparin. HUVECs were seeded on a 96-well plate (coaster3595) at 2  103 cells/ well, and HA-bFGF or bFGF was added to the HuMedia EG-2 without hFGF-b. The cells were incubated at 37 °C in a humidified atmosphere of 95% air and 5% CO2. The culture medium was changed every 2 days. The day on which proteins were added was defined as day 0. The cell growth was observed every day under microscopy, and the numbers of cells on days 1, 3, 5, and 7 were estimated, using the Cell Counting Kit (CCK-8) (DOJINDO). The activity was evaluated at 450 nm as described in the CCK-8 kit. Cell Adhesive Activity. Cell adhesion assays were performed in a 96-well plate (costor3361). The surfaces of the wells were coated with 10 nM of ERE-helix protein (ERE-HB). A fibronectin-coated surface was used as a positive control. BSA-coated and noncoated surfaces were used as negative controls. After incubation for 3 h at 37 °C, each well was washed with PBS, and then the wells were blocked with a 0.1% BSA solution for 2 h at 37 °C. HUVECs, prepared in a HuMedia-EG2 medium at 104 cells/well, were seeded in each plate. After 4 h of incubation at 37 °C, wells were washed with HuMedia-EG2. The remaining number of adhesive cells on the plate was examined using a CCK8 kit. Protein Binding Through a Coiled-Coil Structure. The surface of the wells of a 96-well plate (coster3361) was coated with 100 nM of ERE-HB and incubated for 3 h at 37 °C. After blocking with 0.1% BSA, various concentrations (10 nM, 100 nM, and 1 μM) of HA-FGF (100 μL) were added and the plates were incubated for 3 h at 37 °C. A solution of 100 μL of 1/5000 diluted rabbit anti-bFGF antibody (SIGMA) was reacted for 40 min at 30 °C. Finally, peroxidase-labeled antirabbit IgG antibody was reacted for 40 min at 30 °C. After thorough washing with PBS-T, HRP substrate (KPL) was added and the reaction was stopped after 2 min by adding 1 N HCl, then absorbance at 450 nm was measured using a microplate reader. 2039

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Figure 2. Cell growth with the soluble forms of the proteins. The cell growth activities of cultures with HA-FGF (2), with bFGF (b), and without bFGF (9) were investigated by adding each protein to the HUVEC culture medium.

Growth Factor Activity of Adsorbed bFGF. The surface of wells of a 96-well plate was modified with HA-FGF by attaching a coiled-coil structure with ERE-HB as described in the previous section. HUVECs suspended in HuMedia-EG2 without bFGF at 2.0  103 cells/well were seeded on the plate. The culture medium was changed every 2 days. The numbers of cells on each day were counted using the CCK-8 kit. The growth kinetics of the remaining attached cells were followed for 5 days.

’ RESULTS AND DISCUSSION Expression and Purification of Proteins. E. coli KRX was used as the host strain for the expression of the HA-bFGF and the ERE-HB thioredoxin fusion proteins. In our previous work, bFGF with a His-tag at the N-terminus was expressed in an insoluble fraction, and it had no bFGF activity even after refolding. Therefore, HA-bFGF was expressed as a fusion with thioredoxin at its N-terminus. LaVallie et al. reported that the fusion to thioredoxin increases the solubility of heterologous proteins synthesized in the E. coli cytoplasm, and that thioredoxin fusion proteins usually accumulate to high levels.21 Also, in the case of expression of ERE-HB, thioredoxin was chosen as a fusion partner because the ERE-HB contains hydrophobic domains.20 These fusion proteins with an attached helical peptide region were expressed at high levels in both the soluble and insoluble fractions. The proteins in the soluble fraction could be purified in a single step using a TALON metal affinity gel followed by the digestion with enterokinase to remove the His-tag and thioredoxin moieties. The expected sizes from their amino acid sequences of the HA-FGF and ERE-HB proteins after digestion with enterokinase are 21.1 kDa and 19.5 kDa, respectively. The estimated molecular weights of the proteins from SDS-PAGE (SI Figure S2) were a little larger than the expected sizes, but acidic protein10 and elastin-like polypeptide22 sometimes appeared larger than the calculated molecular weight in SDS-PAGE. Since the proteins bound to columns specifically and they were digested with enterokinase, we used them as objective proteins for the next experiments. Growth Factor Activity of the Free Form of bFGF. Growth factor activities of HA-FGF and bFGF were investigated by adding 20 nM of each protein to the HUVEC culture medium. HUVECs are known to require the addition of bFGF as an essential factor for proliferation in low serum culture conditions. The growth curves of HUVEC with HA-FGF or bFGF are shown in Figure 2. HUVECs grew well in the presence of bFGF or

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Figure 3. Cell adhesion activity of ERE-HB. Each protein was coated on a 96-well plate surface and HUVECs were then seeded. After 4 h of incubation at 37 °C, each well was washed and the remaining numbers of cells were determined. The positive control was fibronectin and samples were represented as the ratio of the number of cells attached to fibronectin-coated wells.

HA-FGF, while the same cells did not grow at all without bFGF. Increased cell numbers were observed in culture with HA-FGF as well as bFGF. These results indicate that HA-FGF retained the cell growth activity of bFGF even after fusion with helix A. Cell Adhesion Activity of ERE-HB. The HUVECs were seeded on ERE-HB-coated, fibronectin-coated, BSA-coated, and noncoated cell culture plates (Figure 3). After 4 h of incubation, the numbers of cells were counted in each plate. Cells were attached to the wells and spread on the fibronectin-coated plate, which was the positive control. However, cell attachment to the wells was barely observed on a BSA-coated plate or a noncoated plate (negative controls). In contrast, cells attached readily to the ERE-HB-coated plate (prepared with a coating concentration of 10 nM ERE-HB) as well as to a fibronectin-coated plate. Indeed, cells do not generally adhere to noncoated and BSA-coated plates, so the observed enhancement of cell adhesion to the ERE-HB-coated plates indicated that the ERE-HB protein itself must have adhered to the plate. The ERE protein adsorption to the plate surface was thus assumed but not examined further, given that this has already been reported elsewhere.20 Because of the strong hydrophobicity of the repeated APGVGV sequence, the ERE adsorbed well onto a hydrophobic plate surface via hydrophobic bonding interactions. Even in this adsorbed state on the solid-phase surface, relatively hydrophilic regions of ERE-HB, such as the RGD sequence and the helix B sequence that was fused with an extremely hydrophilic sequence, retain their structure. The cell attachment on the EREHB-coated plate, according to our previous work,20 should be attributed to the RGD sequence in ERE-HB. Binding of HA-FGF Through Coiled-Coil Structure Formation with ERE-HB. Various concentrations of the HA-FGF were bound to the 96-well plate whose surface was coated with 100 nM of ERE-HB. The amounts of HA-FGF on the surface were indicated using an anti-bFGF antibody (rabbit) and HRP-labeled antirabbit IgG. As shown in Figure 4A, the amounts of HA-FGF bound on the surface increased in a concentration-dependent manner. However, the possibility of nonspecific adsorption of HA-FGF should be considered. To confirm the specific binding between HA-FGF and ERE-HB, ERE and bFGF without the attached helix domains were used for control experiments. In these experiments, 100 nM HA-FGF or bFGF was reacted with the surface coated with ERE-HB or ERE. The FGF moiety was detected as described above using an anti-FGF antibody, and the result is shown in Figure 4B. Among the four combinations 2040

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Figure 5. Competitive inhibition of binding of HA-FGF to ERE-HB using helixA and helix B peptides. The HA-FGF solution was mixed with various concentrations of helix A or helix B peptide, and the mixture was reacted to the ERE-HB-coated plate surface. The amounts of HA-FGF bound on the surface were determined using anti-bFGF antibody (rabbit) with an HRP-labeled antirabbit IgG secondary antibody.

Figure 4. Binding of HA-FGF to ERE-HB with a coiled-coil structure on the solid-phase surface. (a) Various concentrations of HA-FGF were reacted with ERE-HB adsorbed on the 96-well plate surface, and the amounts of bound HA-FGF were determined using anti-bFGF antibody (rabbit) with an HRP-labeled antirabbit IgG secondary antibody. (b) The HA-FGF or bFGF was added to the surface coated with ERE-HB or ERE, and the amounts of immobilized bFGF moiety on each well were determined using an anti-bFGF antibody (rabbit) with an HRP-labeled antirabbit IgG secondary antibody.

tested, only the combination of HA-FGF and ERE-HB showed increased binding of bFGF. This indicates that the binding of HA-FGF to ERE-HB was specific. The FGF binding levels detected using the other three combinations were similar, and they should be considered background signals due to nonspecific adsorption. In order to verify that the binding between HA-FGF and EREHB was due to the formation of a coiled-coil structure between helix A and helix B fused with their respective proteins, competitive inhibition of the binding of HA-FGF in the presence of helix A or helix B peptide was performed. A 100 nM HA-FGF solution was mixed with various concentrations (1 nM to 10 μM) of helix A or helix B, and the mixture was reacted with the EREHB-coated plate surface. The amounts of HA-FGF bound to the surface were determined as described above. The absorbance based on the bFGF surface signal was plotted against the concentration of helix A or helix B (Figure 5). The binding of bFGF was inhibited by addition of either helix A or helix B peptide in a concentration-dependent manner. These results indicated that the binding between HA-FGF and ERE-HB was due to formation of a coiled-coil structure between helix A and helix B from each protein. bFGF Activity on the Plate Surface. HUVECs were seeded on the plate whose surface was modified with HA-FGF through coated-ERE-HB, and their growth curves were measured (Figure 6). On the ERE-HB-coated plate without HA-FGF, which was used as a negative control, clear cell growth was not observed (data not shown). As a control experiment, 100 nM

Figure 6. Cell growth with immobilized HA-bFGF. HUVECs were seeded on the plate whose surface was modified with HA-FGF through coated-ERE-HB (2). bFGF without helix A peptide (b) or only buffer (9) was added to the culture medium at day 0 as a control experiment.

b-FGF without the attached helical peptide was added to the culture medium for seeding HUVECs. In this case, the growth curve was almost the same as that seen in the experiment without bFGF. A little growth was observed at 3 days of culture, but no cell growth was observed after that. Since the culture medium was exchanged at 2 days, bFGF that did not bind to ERE-HB should have been washed out. On the other hand, significant cell growth was observed when HA-FGF was immobilized on the ERE-HB-coated surface. The cells still grew well at 5 days of culture. These results suggest that the FGF moiety in the HA-FGF remained active even after immobilization on the plate surface, presumably through forming a coiled-coil structure with the ERE-HB. The effect of bFGF was sustained for a longer time period because the bFGF was retained in the matrix due to noncovalent bonding.

’ CONCLUSION In this study, a novel method for construction of biomaterials for tissue engineering was developed. A designed extracellular matrix (ERE) and bFGF were combined with a noncovalent bonding by formation of coiled-coil structure. The resulting complex showed good cell adhesive and proliferation activities. One advantage of this technique is that the growth factor or a matrix can be changed depending on the kind of target cell being studied. Preliminary experiments using a system that connected helix B with epidermal growth factor (EGF) yielded similar 2041

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’ ASSOCIATED CONTENT

bS

Supporting Information. Additional information as described in the text. This material is available free of charge via the Internet at http://pubs.acs.org.

’ AUTHOR INFORMATION Corresponding Author

*Phone: +81-45-924-5760. Fax: +81-45-924-5779. E-mail: [email protected].

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