Synthesis and Characterization of Cell-Adhesive Silk-Like Proteins

Feb 24, 2009 - Tetsuo Asakura , Yu Suzuki , Aya Nagano , David Knight ... Yu Suzuki , Rui Takahashi , Tadashi Shimizu , Masataka Tansho , Kazuo Yamauc...
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Biomacromolecules 2009, 10, 923–928

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Synthesis and Characterization of Cell-Adhesive Silk-Like Proteins Constructed from the Sequences of Anaphe Silk Fibroin and Fibronectin Chikako Tanaka†,‡ and Tetsuo Asakura*,† Department of Biotechnology, Tokyo University of Agriculture and Technology, Koganei, Tokyo 184-8588, Japan, and Department of Applied Chemistry, National Defense Academy, Yokosuka, Kanagawa 239-8686, Japan Received December 11, 2008; Revised Manuscript Received January 22, 2009

New silk-like recombinant proteins, [(AAG)6ASTGRGDSPAAS]n and [(AG)9ASTGRGDSPAAS]n, with high cell adhesive activities were designed and produced from E. coli. These are recombinant proteins with characteristic sequences from the silk fibroin of a wild silkworm, Anaphe, and the cell adhesive region, including the sequence RGD derived from fibronectin. They showed higher cell adhesion activity than the parent protein, Anaphe silk fibroin without the RGD sequence. In addition, the activities were very similar to that of collagen, which acted as a positive control. Thus, it is demonstrated that the primary structure of Anaphe silk fibroin, which is composed largely of alanine and glycine residues, can be used as a platform for the basic structures of silk-like cell adhesive proteins. The structural characterization of the silk-like recombinant proteins was performed with 13C CP/MAS NMR.

Introduction Silks are generally defined as fibrous proteins that are spun into fibers by some lepidoptera larvae such as silkworms, spiders, scorpions, mites and flies.1 Silk fibroin from the domesticated silkmoth, Bombyx mori, has been studied extensively because of its excellent mechanical properties environmental stability, and biocompatibility which makes it suitable for biomaterials2 such as sutures, artificial skins,3 burn wound dressings,3 artificial bone matrices,4 and artificial tendons.5 The silk fibroin can be described as a block copolymer containing crystalline domains (regions of the polypeptide containing short side-chain amino acids), interrupted with amorphous domains consisting of the bulkier amino acids. Approximately 70% of the fibroin is composed of the amino acid sequence, GAGAGS. A more extended sequence has been reported for the crystalline domain precipitated after chymotrypsin hydrolysis, as GAGAGSGAA[SGAGAG]8Y.6 This sequence accounts for 55% of fibroin. Other characteristics of the primary structure in B. mori silk fibroin are repeating motifs such as AGYGAG and AGVGYGAG as well as the irregular unit, GAAS.7,8 On the other hand, there are a variety of silk fibroins from wild silkworms, which have different primary structures from B. mori silk fibroin although the main amino acid residues are still G and A. We have reported structural analyses of silk fibroins from wild silkworms such as Samia cynthia ricini, Antheraera pernyi, and spider dragline silks using NMR methods.9-16 The presence of a repeated polyalanine region is characteristic of the primary structures of the latter silk fibroins. Recent progress in biotechnology makes it possible to prepare several silk-like proteins by combining the characteristic sequences extracted from silk fibroins of several silkworms and spider silks.17-21 In addition, for example, introduction of the * To whom correspondence should be addressed. Tel./Fax: +81-42-3837733. E-mail: [email protected]. † Tokyo University of Agriculture and Technology. ‡ National Defense Academy.

sequence RGD from fibronectin with cell adhesive character into silk-like proteins increases the cell adhesive ability of the silk-like recombinant proteins remarkably, which are then more suitable for biomaterials.19,22 In our previous paper,23 we focused on silk fibroin from a wild silkworm, Anaphe, which is highly abundant in equatorial and southern Africa.24,25 The silk fibroin fiber has a high strength and toughness, similar to that of B. mori.26 However, the amino acid composition of the silk fibroin from Anaphe is much simpler than that of B. mori.27-29 The Ala content is very high, 60 mol %, and the Gly content is more than 30 mol %. Thus, the sum of Ala and Gly content accounts for more than 90 mol %. Therefore, Anaphe silk fibroin seems to be one of the best candidates to design fibroin-mimetic novel recombinant protein. The main sequences of the silk fibroin were mixtures of the sequences, (AG)n1 and (AAG)n2.23 These sequences look very similar to those observed in the minor ampullate silks produced by orb-spiders.30 In this paper, we designed new silk-like recombinant proteins, [(AAG)6ASTGRGDSPAAS]n and [(AG)9ASTGRGDSPAAS]n for biomaterials with high cell adhesive ability. These are recombinant proteins composed of the characteristic sequences from Anaphe silk fibroin and the cell adhesive region including the sequence RGD derived from fibronectin. This RGD unit has been reported to lie on a conformationally mobile loop in fibronectin.31 The cell adhesive sequence, TGRGDSPA, has been reported to retain higher activity than RGD or RGDS alone when incorporated into human lysozyme.32 In addition, we have reported several cell adhesive silk-like proteins by introducing the sequence TGRGDSPA from fibronectin into several fundamental sequences from silk fibroins.19,22 The lengths of these silk-like sequences in the designed proteins were determined by reference to the recombinant cell adhesive silk-like proteins reported previously.18,22 Namely, the sequence (GAGAGS)3 was selected; 18 residues that consist of three times repeating of GAGAGS. Thus, n1 and n2 values in the sequences, (AG)n1 and (AAG)n2 were decided to be 9 and 6, respectively. It is

10.1021/bm801439t CCC: $40.75  2009 American Chemical Society Published on Web 02/24/2009

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Figure 1. Designed oligonucleotide sequences for Anaphe silk-like hybrid proteins, AAGFN10 and AGFN10. (a) Oligonucleotide sequences of synthetic adapter inserted into pUC118 to create pUC118-linker, (b) Oligonucleotide sequences of cell adhesive sequence block; FN, (c) and (d) oligonucleotide sequences of Anaphe silk fibroin block I (AAG) and II (AG), used for cloning of the hybrid proteins.

expected that these silk-like sequences form similar secondary structure as silk fibroins by hydrophobic interactions. The genes encoding the recombinant proteins were constructed and expressed in Escherichia coli. The structural characterization of the recombinant proteins was performed with 13C CP/MAS NMR, and cell adhesion and cell culture assays were performed.

Materials and Methods Materials. Plasmids pUC118 and pET30a were obtained from Takara Bio. Inc. and Novagen, respectively. E. coli DH5R was used for propagation and construction of plasmids, and BL21(DE3)pLysS was used for expression of proteins. The synthesis of the oligonucleotides designed in this study was performed by Asahi Techno Glass Corporation (Tokyo, Japan). Restriction and ligase enzymes were purchased from Takara Bio Inc. (Shiga, Japan). Normal human dermal fibroblasts (NHDF) were obtained from Kurabo Co. Ltd. (Osaka, Japan). Production of Silk-Like Recombinant Proteins. The duplex DNA shown in Figure 1a was ligated into pUC118 to construct pUC118linker vector, which provides SpeI and NheI restriction enzyme sites for the polymerization of target DNA. The other three duplex DNAs listed in Figure 1b-d encode the cell adhesive sequence from fibronectin (FN) and the main motifs of Anaphe silk fibroin (AAG and AG), respectively. These oligonucleotide fragments contained different restriction enzyme sites at the 5′- and 3′-termini to control the sequence orientation resulting from ligation. Therefore, the monomers of AAGFN and AGFN were constructed by ligating the oligonucleotides (AAG and FN or AG and FN) and inserted into BamHI-digested pUC118. After digestion of the recombinant pUC118 containing a monomer fragment with NheI and SpeI, the fragment was ligated into the NheISpeI site of pUC118-linker. The multimers of AAGFN and AGFN were obtained by using previously reported strategies involving head-to-tail ligation and orientation for NheI and SpeI sites.19,20,22,33 For the expression, the multimerized DNA fragments were subcloned into pET30a (Novagen Inc.) by BamHI and HindIII and transformed into BL21(DE3)pLysS. Large scale cell cultivations were performed in Terrific Broth (TB) containing 25 µg/mL of chloramphenicol and kanamycin at 30 °C. Protein expression was induced by addition of IPTG to final concentration of 1 mM after reaching an OD600 between 0.6 and 0.7. The expressions were continued for 1 h. After collection of cells, the cells were resuspended in the lysis buffer (8 M urea, 100 mM NaH2PO4, 10 mM Tris, pH 8.0) at 5 mL per gram of wet weight of cell pellet, and cells were disrupted by supersonication. Cellular debris was removed by centrifugation (14500 rpm, 30 min, 4 °C), and the supernatant was loaded onto a nickel chelate affinity column to purify the fusion proteins by using a His-tag. The column was washed

with lysis buffer and wash buffer (8 M urea, 100 mM NaH2PO4, 10 mM Tris, pH 6.3). The protein was eluted with elution buffer (8 M Urea, 100 mM NaH2PO4, 10 mM Tris, pH 4.2). The eluted protein was dialyzed against distilled water for 4 days and then lyophilized. SDS-PAGE gels were stained with Coomassie Brilliant Blue R-250. The western-blottings were performed by standard semidry transfer method and an antibody for His-Tag was used. Solid State 13C CP/MAS NMR. The silk-like hybrid proteins, AGFN10 and AAGFN10, were dissolved in FA (formic acid) or TFA (trifluoroacetic acid). The solutions were dried under nitrogen gas at room temperature. Similarly, a mixture of these two recombinant proteins, in a 2:3 mixture ratio of AGFN10 and AAGFN10, was prepared from TFA and FA solutions. A dialyzed sample of the mixture was prepared from 60% (w/w) LiSCN solution, dialyzed against distilled water for 4 days and then lyophilized. The model peptides, 13C(AG) 7 FN: (AG) 3 [3- 13 C]AG(AG) 3 AST[2- 13 C]GR[1- 13 C]GDSPAAS(AG)7 and 13C-(AAG)5FN: (AAG)2A[3-13C]AG(AAG)2AST[213 C]GR[1-13C]GAS(AAG)5 were synthesized as described in a previous paper34 and soaked in water overnight after FA treatment. Solid state 13 C CP/MAS NMR spectra were observed on a Chemagnetics CMX400 spectrometer operating at 100 MHz, with a CP contact time of 1 ms, TPPM decoupling, a spectral width of 60 kHz, recycle delay of 5 s, and magic angle spinning at 7 kHz. Chemical shifts are reported relative to TMS as reference. Cell Adhesion Assay on Protein-Coated Plates. Recombinant proteins and Anaphe silk fibroin were dissolved in FA at a concentration of 300 µg/ml. The solutions were added to 96-well microplates, 25 µL/well. After drying at room temperature overnight, the plates were desiccated under vacuum conditions for 2 h. Collagen I used for a positive control was diluted to 150 µg/mL by HCl aqueous solution and added to 96-well microplates, 50 µL/well. After incubation for 1 h, the solutions were removed. All of the surfaces were washed with PBS and distilled water twice each, and then saturated with 1 mg/mL of BSA for 2 h. Wells were washed with PBS and distilled water twice each, and filled with 50 µL of serum-free DMEM medium. The plates were kept in a 37 °C incubator for 1 h. NHDF cells (Neonatal Normal Human Dermal Fibroblasts) were used for the cell adhesion assay. Cells peeled from plates were washed and resuspended into serum-free DMEM (2.0 × 105 cells/mL). Cell suspension (50 µL) was added to protein-coated plates that had been kept in the incubator, so that the number of cells was 1.0 × 104, and the total volume of medium was 100 µL per well. After incubation for 3 h at 37 °C, 5% CO2, the number of cells was counted by a MTS assay Kit (Promega).

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Figure 2. SpeI and NheI digestion analyses of multimerized genes encoding AAGFN (a) and AGFN (b) on cloning vectors. Lane 1, 100 bp markers; lane 2, pUC118-linker-monomer; lane 3, pUC118-linkerdimer; lane 4, pUC118-linker-trimer; lane 5, pUC118-linker-pentamer; and lane 6, decamer.

Results and Discussion Production of Silk-Like Proteins. Figure 2 shows the results of 1.5% agarose gel electrophoresis of pUC118-AAGFN (a) and pUC118-AGFN (b) after digestion with NheI and SpeI, corresponding to the construction of 1, 2, 3, 5 and 10 repeats of the monomer DNA. Both the monomer genes of AAGFN and AGFN are 86 bps each, as shown in Figure 1. The monomer bands were observed at a little smaller than the 100 bps marker band. The theoretical length of the multimers; dimer, trimer, pentamer and decamer, after digestion with NheI and SpeI are 176 bps, 266 bps, 446 bps and 896 bps and 846 bps, respectively. Therefore, it is concluded that these two kinds of silk-like protein genes were both successfully constructed. All of these DNA sequences were confirmed by sequencing. The purified 10 repeated DNA fragments were inserted into the commercially available expression plasmid pET30a, which contains a [His]6 sequence at the N-terminus and C-terminus of the silk-like proteins for purification by immobilized metal affinity chromatography. The plasmid vectors encoding silklike proteins AAGFN10 and AGFN10 were transformed into BL21(DE3)pLysS and the encoded proteins were expressed upon induction by IPTG. The proteins were purified by NiNTA affinity chromatography using urea buffers. The results of SDS-PAGE gel and Western blotting for the purified Histagged silk-like proteins AAGFN10 and AGFN10 are shown in Figure 3. The purity of the silk-like proteins was checked by the presence of a single band on SDS-PAGE. The molecular weights of AAGFN10 and AGFN10 are about 30.6 and 30.2 kDa, respectively, which was in agreement with the molecular weights estimated from SDS-PAGE. The yield was about 20 mg/L for each recombinant protein. Structural Characterization of Recombinant Proteins in the Solid State. The 13C CP/MAS NMR spectra of AAGFN10 after dialysis (a) TFA (b) and FA (c) treatments are shown in Figure 4. The 13C CP/MAS NMR spectra of AGFN10 are shown in Figure 5 in the same way. These treatments induced conformational changes of their model peptides.34 The FA treatment is known to induce β-sheet formation into silk fibroin and model peptides.35 The secondary structure of all silk fibroins in the fibrous state is β-sheet. Therefore, β-sheet formation is considered to be essential to silk-like sequences for using as materials. In addition, dialysis against distilled water is known to induce the formation of silk I conformation in B. mori silk fibroins and their model peptides,36 while TFA induces the formation of R-helix structure, which is the secondary structure of S. c. ricini silk fibroin before spinning, to a model peptide of S. c. ricini silk fibroin.11,12,37 Therefore, these treatments are considered to be useful to evaluate the structural properties as

Figure 3. The results of SDS-PAGE (top) and Western blotting (bottom) of the successful construction and purification of the recombinant proteins, AAGFN10 (a) and AGFN10 (b). Lane M, molecular weight markers; lane U, unrefined solution; lane F, flow through fraction of the column; lane L, lysis buffer fraction; lane W, wash buffer fraction; and lane E, elution buffer fraction.

Figure 4. 13C CP/MAS NMR spectra of AAGFN10 after dialysis (a), TFA treatment (b), and FA treatment (c).

silk-like proteins, although we do not know the secondary structure of Anaphe silk fibroin before spinning. All the spectra include broad peaks, which are considered to derive from a random coil conformation of the cell adhesive sequence parts, because the recombinant proteins did not include a site-specific 13 C-label. However, the chemical shifts of the main peaks from Ala and Gly residues can be used to indicate the secondary structure of silk-like regions. The chemical shift values and the secondary structures of silk-like regions were summarized in Table 1. After FA treatment, AAGFN10 (Figure 4c) and AGFN10 (Figure 5c) displayed β-sheet structure in the same way as other silk-like proteins and silks. This is also the same secondary structure as the Anaphe silk fibroin fiber. It suggests that these recombinant proteins will be insoluble in water after FA treatment. On the other hand, the recombinant proteins, AAG-

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Figure 5. 13C CP/MAS NMR spectra of AGFN10 after dialysis (a), TFA treatment (b), and FA treatment (c).

Figure 6. 13C CP/MAS NMR spectra of a mixture of the recombinant proteins, AAGFN10 and AGFN10 in a proportion of 3:2, after dialysis (a), TFA treatment (b), and FA treatment (c).

FN10 and AGFN10, showed different characteristic spectra after dialysis and after TFA treatment. For AAGFN10, the silk-like part of the sequence, (AAG)6, formed typical R-helix structure after TFA treatment (Figure 4b), while after dialysis, it was a mixture of random coil and R-helix (Figure 4a). This means that the (AAG)6 sequence has a tendency to form R-helix, similar to polyalanine. As for AGFN10, the silk-like part of the sequence, (AG)9, was random coil after TFA treatment (Figure 5b), whereas it took a silk I conformation after dialysis (Figure 5a). This suggests that the silk-like sequence region can make crystalline domains because the formation of silk I is achieved by alternate inter- and intramolecular hydrogen bonds.36 The 13 C CP/MAS NMR spectra of a mixture of these two recombinant proteins, AAGFN10 and AGFN10 in a proportion of three to two, are shown in the same manner as the others in Figure 6 because the primary structure of silk fibroin from Anaphe includes both motifs, (AAG)n1 and (AG)n2, in a similar ratio, presumably. As expected, the mixture of these proteins also formed β-sheet structure after FA treatment similar to the others (Figure 6c). The silk-like sequences were random coil after dialysis (Figure 6a), while they were a mixture of R-helix and β-sheet after TFA treatment (Figure 6b). These structures suggest that the original structures do not form independently,

because the R-helix structure formed by the (AAG)6 sequence and the silk I conformation formed by the (AG)9 sequence were not formed in the mixture. That is, mixing with other sequences can cause a structural change as well as combining the sequence in the same chain. These structural behaviors can be considered to be available for the control of solubility in water. Cell Adhesion Activity of AAGFN10 and AGFN10. The number of adhered NHDF cells was counted by using a MTS assay for each protein coated plate, using the FA solution. If the recombinant proteins are to be used as biomaterials, they will be exposed to water because cells need a liquid medium. Under such a condition, the silk-like sequence should form a β-sheet structure to avoid solubilization in water. In fact, the silk fibroins change the solubility character in water from soluble to insoluble by the structural change of their crystalline regions to β-sheet. Therefore, it is reasonable to check the cell adhesive activities of the recombinant proteins under conditions where the secondary structure of the silk-like sequence part was β-sheet. FA was chosen as a coating solvent because the silklike sequence parts of the recombinant proteins formed β-sheet after FA treatment (Figure 4c and 5c). The Anaphe fibroin was used for this assay as a negative control because it is expected

Table 1. 13C Chemical Shifts (in ppm from TMS) of Recombinant Proteins after each Treatment and the Secondary Structures of the Silk-Like Regions 13

C chemical shift

sample AAGFN10 AGFN10 mixture (AAGFN10andAGFN10)

treatment dialysis TFA FA dialysis TFA FA dialysis TFA FA

Ala Cβ 15.8, 15.0 15.6, 16.7 15.6 16.2, 16.4 15.4, 16.5,

19.5 19.9 20.5 20.1 20.1

Ala CR

Gly CR

49.3, 52.3 52.6 48.7, 51.6 50.9 50.8 48.9 51.5 49.1, 52.3 49.2

42.4 44.6 42.1 43.0 42.0 42.5 43.3 42.6 42.5

conformation of the sequence from silk fibroin R-helix and random coil R-helix β-sheet and random coil silk I random coil β-sheet random coil R-helix and β-sheet β-sheet

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the combined cell adhesion sequence worked successfully in both recombinant proteins.

Conclusions

Figure 7. Histograms of the relative cell adhesion activities of silklike hybrid proteins coated on a plate with FA. The activity of collagen, Anaphe silk fibroin and nontreatment were also shown for positive and negative controls.

Figure 8. 13C CP/MAS NMR spectra of model peptides, 13C-(AAG)5FN (a) and 13C-(AG)7FN (b), under wet (black lines) and dry (gray lines) conditions after FA treatment.

not to have an active RGD sequence and the recombinant proteins were designed based on the primary structure. The observed cell adhesion activities are shown in Figure 7, as histograms of relative activities compared to the nontreatment plate as a standard. The Anaphe silk fibroin showed higher cell adhesion activity than that of the noncoating standard, which suggests that the Anaphe fibroin has no toxicity for cells, in the same way as B. mori silk fibroin, which has been widely used as a biomaterial. The hybrid proteins showed higher activities than the parent protein, Anaphe silk fibroin without the RGD sequence, as expected. In addition, the activities were very similar to that of collagen, the positive control. Dynamics of Model Peptides. The proteins were exposed to liquid medium during the cell adhesion assay. Therefore, to consider the effect of the presence of water, a 13C CP/MAS NMR measurement for the 13C-labeled model peptides was performed under wet conditions (Figure 8). The spectra measured under dry conditions are shown in gray lines for comparison. The relative intensities of the 13C-labeled peaks from the glycine residues decreased in the spectra measured under wet conditions in both of the spectra, 13C-(AAG)5FN (Figure 8a) and 13C-(AG)7FN (Figure 8b). On the other hand, the relative intensities of the alanine Cβ peaks were not changed by immersion in water. The reason for the decrease in the intensities of these peaks is suggested to be due to the reduction of the efficiency of cross-polarization by the effect of increasing the local mobility at these regions. The 13C-labeled glycine residues are located in the cell adhesive region; to the contrary, the 13C-labeled alanine residue is located in the center of silklike region. Therefore, it is concluded that under the conditions where the cell adhesion activities were assayed, the cell adhesive regions keep a conformationally mobile state, which is corresponding to the original motional state in fibronectin, while the silk-like regions keep rigid β-sheet state. By considering also the results of the cell adhesion assay, it can be concluded that

Novel silk-like recombinant hybrid proteins, [(AAG)6ASTGRGDSPAAS]n and [(AG)9ASTGRGDSPAAS]n, which combined Anaphe silk-like sequences with a cell adhesive sequence derived from the III10 module in fibronectin, were designed and successfully produced. The silk-like sequence region of these recombinant proteins took β-sheet structure, which is the secondary structure of silk fibroin from Anaphe in the fibrous state with good mechanical properties, after FA treatment. In contrast, other treatments induced structural change to the silk-like sequence parts. These structural behaviors are one of the characters of silk fibroins. Moreover, using a cell adhesion assay and the 13C CP/MAS NMR measurements for the model peptides under wet conditions, it was concluded that the hybridized cell adhesion sequence worked in these recombinant proteins. Therefore, these hybrid proteins created here will be strong candidates for a successor to B. mori silk-like biomaterials. Thus, it was indicated that the primary structure of Anaphe silk fibroin, which is composed largely of alanine and glycine residues, can be used as a platform for the basic structures of silk-like cell adhesive proteins. The obtained knowledge in this research will underlie the development of silk-like proteins for tissue engineering. Acknowledgment. T.A. acknowledges support from Promotion of Basic Research Activities for Innovative Biosciences, Japan. We also acknowledge Prof. Mike Williamson at University of Sheffield for many suggestions and comments.

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