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Jan 30, 2018 - of genome editing technology to the structural study of silk. The origin of a ..... Noboru OHTA (JASRI, Japan) for his technical suppor...
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Letter Cite This: ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

Genome Editing Advances the Structural Study of Silk Taiyo Yoshioka,† Yoko Takasu,† Hideki Sezutsu,‡ and Tsunenori Kameda*,† †

Silk Materials Research Unit and ‡Transgenic Silkworm Research Unit, National Agriculture and Food Research Organization (NARO), 1-2 Ohwashi, Tsukuba, Ibaraki 305-8634, Japan S Supporting Information *

ABSTRACT: We first applied the genome edited silkworm silk (GE-silk) to interpret X-ray fiber diagram, and implied a great potential for the application of genome editing technology to the structural study of silk. The origin of a weak meridional layer-line streak with a spacing of ∼21 Å, observed in the Xray fiber diagram of Bombyx mori silkworm silk, has been widely believed but not experimentally proven to be a period of the pseudostructure associated with the occurrence of serine residues at regular intervals in a hexapeptide repeating unit -G-A-G-A-G-S-. The above hypothesis was experimentally demonstrated from X-ray measurements of GE-silk.

KEYWORDS: Bombyx mori silkworm silk, silk fibroin, silk crystal, β-sheet, amino acid sequence, gene modification, TALENs, synchrotron X-ray analysis Gly)) as the first approximation to their structural models. In fact, this -G-A- sequence approximately fits well into the fiber period of 6.98 Å estimated from the X-ray fiber diagram. On the contrary, apart from the fiber period of 6.98 Å, a weak meridional layer-line streak of spacing around 21 Å, which is almost triple the length of the fiber period, was also observed.5,6,8 This streak was reasonably attributed to a period of the pseudostructure associated with the occurrence of serine residues at regular intervals in the hexapeptide repeating unit of -G-A-G-A-G-S-.6,8 However, it has not been proved experimentally until now. To investigate the origin of this layer-line streak, here, structural analysis of a GE-silk fiber, in which the ratio of the repeated sequence of -G-A-G-A-G-S- to the total amino acid residue was significantly reduced, was performed. The results of this study would support the finding that the layer-line streak is certainly attributed to the regular intervals of serine residues in the repeated sequence. Silk-fibroin protein consists of heavy (H) and light (L) chain polypeptides of ∼350 and 26 kDa, respectively, linked by a disulfide bond at their C-terminus.9,10 The entire amino acid sequence of the H-chain was determined by Zhou et al. in 2000 and was revealed to be composed of 12 repetitive and 11 intervening sequence domains.11 The former and latter domains are often called crystalline and amorphous domains, respectively.12 Recent progress in genome editing technology13 has enabled editing of any endogenous gene in the silkworm genome with high efficiency.14−17 Figure 1a schematically explains the

tructural study of the native silk fiber produced by the silkworm Bombyx mori has been conducted extensively for a long time to gain a deeper understanding about the origin of its superior mechanical property. In a historical fact, the invention of an analytical technique largely advances the understanding of the structure of silk.1 This technical invention includes not only the improvements in analytical instruments and structure solving algorithms and software, but also the advances in sample preparation techniques. In this study, a novel sample preparation technique for X-ray diffraction analysis of silk fiber is proposed. The protein sample is molecular-designed by modifying silk amino acid sequence. This designed silk-like protein is biosynthesized and spun into the silk fiber by silkworm genetically modified by genome editing technology. This study emphasizes that the silk fibroin obtained from genome-edited silkworms (GE-silk) makes possible the detailed interpretation of the X-ray fiber diffraction patterns, and has a great potential for the application to the structural study of silk. The X-ray diffraction study on the silk II crystalline modification formed in the silk fiber has been carried out by several research groups.2−6 The most accepted structural model of the silk II form was proposed by Takahashi et al.6 in 1999 to be an “antipolar−antiparallel” type pleated-β-sheet structure with the unit-cell parameters of a = 9.38 Å, b = 9.49 Å, and c (fiber axis) = 6.98 Å and the space group P21-C22 which was based on a “polar−antiparallel type model” previously proposed by Marsh et al.5 Since Lucas et al.7 had revealed that the crystalline region of silk dominantly contains a regular repetition of the amino acid sequence “(-G-A-G-A-G-S-)n”, where G, A, and S denote glycine, alanine, and serine, respectively, both Marsh et al. and Takahashi et al. adopted a repeating unit of “-G-A-” (namely, poly(Ala-

S

© XXXX American Chemical Society

Received: January 12, 2018 Accepted: January 30, 2018 Published: January 30, 2018 A

DOI: 10.1021/acsbiomaterials.8b00043 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

Letter

ACS Biomaterials Science & Engineering

Figure 1. Schematic representation of (a) the preparation scheme of the truncated fibroin H-chain gene encoding GE silk, (b) the resultant GE-silk protein, (c) SDS-PAGE analysis for the native and GE silks, and (d) the TALEN binding to the target region.

preparation of the truncated fibroin H-chain gene encoding GEsilk used in this study (also see Figures S1 and S2). Most of the GA repeat-encoding regions were eliminated using transcription activator-like effector nucleases (TALENs) that specifically cleave the intervening sequences between the GA-rich domainencoding repetitive sequence (the TALEN binding sequences were underlined in Figure 1d), so that the second to 11th repetitive domains in the total 12 regions of the original protein were removed in the resultant modified protein as schematically shown in Figure 1b. The nucleotide sequence including the whole repetitive sequences of thus created GE silk fibroin Hchain gene and the amino acid sequence of the corresponding region, which evidently showed a successful achievement of the aimed genome editing, are shown in Figure S3. The perfect replacement of a native H-chain fibroin by this GE-one was confirmed by SDS-PAGE analysis. While the native fibroin Hchain band was observed at around 350 kDa, the GE mutant fibroin H-chain was detected at around 75 kDa instead (see Figure 1c). It should be emphasized that such a complete replacement of a native protein by a modified protein is the unique point in genome editing technique and has never been achieved in the conventional transgenic method. In the 12 repetitive sequence domains in the native silk, 432 repeating units of -G-A-G-A-G-S- were included, which correspond to ∼49% of the total amino acid residues (see Table 1). This was estimated from the amino acid sequence for the native fibroin H-chain of Bombyx mori silkworm silk reported by Zhou et al.11 (accession no. AF226688). In contrast, in the GE-silk, the total repeating unit was reduced up to 31, corresponding to ∼24% of the total residues (see the amino acid sequence shown in Figure S3). In addition, the distribution of the repeating number “n” in the repeating block “(-G-A-G-AG-S-)n” was changed drastically by genome editing as shown in Figure 2. The crystallite size of D002 along the fiber axis in the native silk fiber has been reported with a dispersion from 100 to 160 Å.18,19 If the crystal of silk II formed in the native silk fiber is

Table 1. Comparison of the Total Numbers of the Amino Acid Residues and the Repeating (GAGAGS)n Sequence Units of n ≥ 1 and n ≥ 5 in the Native and GE Silks total residue native silk GE silk

5242 781

(GAGAGS)n unit, n ≥ 1 (GAGAGS)n unit, n ≥ 5 432 (49%)a 31 (24%)a

250 (29%)a 14 (11%)a

a

The percentage values given in the parentheses are the ratio of the amino acid residue contained in the (GAGAGS)n repeating units to the total amino acid residue.

Figure 2. Counts of the repeating block “(-GAGAGS-)n” having the repeating number “n” in the native and GE silks.

assumed to be composed of only the repeating blocks “(-G-A-GA-G-S-)n”, the repeating number of roughly 5 and 8 are required for the crystallite sizes of D002 to be 100 and 160 Å, respectively. Although ∼29% of the total amino acid residues belong to such a long-range repeating block “n ≥ 5” in the native silk, only ∼11% belongs to that in the GE silk (see Table 1). WAXD 2θ profiles measured for the native and GE-silk fibers are shown in Figure 3a. Both the profiles were assigned to the silk II modification6 (Dominant formation of silk II modification in both silks was confirmed also by the solid-state 13C NMR spectra shown in Figure S4). The degrees of crystallinity for them were B

DOI: 10.1021/acsbiomaterials.8b00043 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

Letter

ACS Biomaterials Science & Engineering

Figure 4. SAXS q-profiles obtained from the native and GE silks.

widely believed to be a period of the pseudostructure associated with the occurrence of serine residues at regular intervals in a hexapeptide repeating unit -G-A-G-A-G-S-. To demonstrate this hypothesis experimentally, we for the first time applied genomeediting technology to the structural study of silk fiber. To reduce the ratio of the repeating unit -G-A-G-A-G-S- to the total amino acid residue in the fibroin H-chain, we successfully removed the second to 11th repetitive domains of the total 12 repetitive domains by using a highly efficient pair of TALENs. As a result, the ratio of the repeating unit to the total amino acid residue was significantly reduced from ∼49% in the case of native silk to ∼24% in GE-silk. Especially, the ratio of the long-range repeating block of (-G-A-G-A-G-S-)n, where n ≥ 5, in the GE-silk was further reduced from ∼29 to only ∼11%. It was confirmed by synchrotron X-ray scattering measurement that such repeatingunit-reduced GE silk does not yield 21 Å scattering in question. This experimental fact strongly supports the above-mentioned hypothesis as to the 21 Å layer-line scattering. This study showed us a great potential for the application of genome editing technology to the structural study of silk fiber having several unsolved subjects.

Figure 3. (a) WAXD 2θ profiles obtained from the native and GE silks, and (b) the results of peak separation analysis for them.

estimated to be ∼47 and ∼40%, respectively, from the WAXD 2θ profiles using peak separation analysis (Figure 3b). Although the values of crystallinity estimated from such limited number of crystalline reflections might be not very accurate, the estimated value for the native silk showed a good agreement with the previously reported values.18,20 Despite the drastic reduction of the repeating sequence “(-G-A-G-A-G-S-)n”, especially, with long-range repeating-sequence of n ≥ 5, the degree of crystallinity of GE-silk decreased only slightly from the native silk. This maintenance of high crystallinity of ∼40%, in spite of such low degree of long-range repeating-sequence (∼11%), strongly indicated that the silk II form of the β-sheet crystal is not only composed of the repeating sequence “(-G-A-G-A-G-S-)n”, but also contains the other sequence combinations as proposed by Zhou et al.12 By the way, the genome editing, conducted here, altered drastically the mechanical property of silk fibers, so that the GE-silk fibers became a quite brittle nature (see Figure S5 and Table S1). Because of the serious brittleness, it was not able to prepare a parallel-aligned fiber-bundle for measuring the WAXD fiber diagram which is necessary for evaluating the crystallite size, D002, along the c-axis direction. For the further detailed discussion about the constituent amino acid sequence in the crystallite of GE-silk, the evaluation of D002 remains as an important future subject. The GE-silk used in this study was designed to have significantly less repeating blocks of “(-G-A-G-A-G-S-)n”, especially with the long-range sequence of n ≥ 5. As mentioned above, the WAXD fiber diagram of Bombyx mori silk shows a weak meridional layer-line streak of the spacing of around 21 Å, reasonably ascribed to the periodicity of regular interval of serine residue in the repeating block “(-G-A-G-A-G-S-)n”. If this assumption is correct, the periodic scattering must be reduced in the GE-silks compared to that in the native silk. To confirm this, X-ray scattering measurements for the native and GE silks were performed using a highly brilliant synchrotron X-ray beam. The one-dimensional q-profiles measured from the native and GE silks are shown in Figure 4, where the scattering vector q is defined as 4π sin θ /λ. Although diffuse scattering, corresponding to the spacing of ∼21 Å, was clearly observed in the native silk, no scattering was detected in the GE-silk. This is the first experimental result supporting the above-mentioned assumption.

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EXPERIMENTAL SECTION

Sample Preparation. To establish a mutant strain producing GE silk, we utilized TALENs that specifically bind to the sequence well conserved in the second to 10th intervening sequence-encoding regions of the fibroin H-chain gene. The recognition sequences of the TALEN pair are 5′-TATGTAGCAAATGGCG-3′ of the sense strand and 5′TGACGACCAAGCGTAT-3′ of the antisense strand of intervening sequence-encoding region (Figure S1). A pair of TALENs was constructed with a Golden Gate TALEN and TAL Effector Kit21 using the pBlue-TAL vector optimized for Bombyx gene targeting.14 TALEN mRNA was in vitro synthesized using mMESSAGE mMACHINE T7 Kit (Ambion) and microinjected to 135 silkworm embryos in the same method as our previous report. 14 The microinjected individuals were reared on artificial diet and the moths were crossed with each other or wild type moths. The G1 silk moths that produced 75 kDa silk protein in the cocoon were selected to establish a mutant strain (Figure S2). Sequencing of the Truncated Fibroin H-Chain Gene. The repetitive part of the truncated fibroin H-chain gene was amplified by genomic PCR using the specific primers to the 5′ region (5′TGCTCAAAGTTATGTTGCTGCTGA-3′) and the 3′ region (5′TAGTCGTAACTGCGAGATGAAGCA-3′) of the gene. The PCR product was subcloned into a pTA2 vector (Toyobo, Osaka, Japan) and then sequenced using M13-20 (5′-GTAAAACGACGGCCAGT-3′) and M13RV primers (5-CAGGAAACAGCTATGAC-3′), and the primer specific to the boundary region of the last intervening and repetitive sequences (5′-CGCTTCCAGTTTCAAAGTCAGATT-3′). X-ray Diffraction. The crystalline modification and degree of crystallinity of the native and GE-silk fibers were evaluated by WAXD analysis using an X-ray diffractometer XRD-6000 (40 kV, 30 mA, CuKα; Shimadzu Co., Japan). The cocoon fibers were cut into small pieces and

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CONCLUSIONS The origin of a weak meridional layer-line streak with a spacing of ∼21 Å, observed in the X-ray fiber diagram of silk fiber, has been C

DOI: 10.1021/acsbiomaterials.8b00043 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

Letter

ACS Biomaterials Science & Engineering set on the sample stage without specific orientation. The degree of crystallinity (Xc) was estimated from the obtained 2θ-profile using the following formula: Xc = Ic/(Ic + Ia) × 100%, where Ic and Ia are the total intensities of the crystalline and amorphous diffraction peaks, respectively. The intensities of the crystalline and amorphous peaks were evaluated by peak separation analysis, conducted using free software Fityk 0.8.6 (developed by Marcin Wojdyr). The contributions of five crystalline reflections (100, 200, 210, 211, and 300 reflections) and one amorphous halo, all of which were fitted by Gaussian distribution, were used for peak separation. The synchrotron X-ray scattering measurements for the cocoon fibers (with random fiber alignment) of native and GE silks were performed using SPring-8 beamline 40B2 (Hyogo, JAPAN). The wavenumber of irradiant X-ray beam (λ) was 0.0709 nm. A sample-to-camera distance was 390 mm, and an imaging plate system of R-Axis VII (Rigaku Co., Japan) was used as a detector.

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(6) Takahashi, Y.; Gehoh, M.; Yuzuriha, K. Structure refinement and diffuse streak scattering of silk (Bombyx mori). Int. J. Biol. Macromol. 1999, 24, 127−138. (7) Lucas, F.; Shaw, J. T. B.; Smith, S. G. The amino acid sequence in a fraction of the fibroin of Bombyx mori. Biochem. J. 1957, 66, 468−479. (8) Fraser, R. D. B.; MacRae, T. P. In Conformation in Fibrous Proteins and Related Synthetic Polypeptides; Academic Press: New York, 1973. (9) Takei, F.; Kikuchi, Y.; Kikuchi, A.; Mizuno, S.; Shimura, K. Further evidence for importance of the subunit combination of silk fibroin in its efficient secretion from the posterior silk gland cells. J. Cell Biol. 1987, 105, 175−180. (10) Tanaka, K.; Kajiyama, N.; Ishikura, K.; Waga, S.; Kikuchi, A.; Ohtomo, K.; Takagi, T.; Mizuno, S. Determination of the site of disulphide linkage between heavy and light chains of silk fibroin produced by Bombyx mori. Biochim. Biophys. Acta, Protein Struct. Mol. Enzymol. 1999, 1432, 92−103. (11) Zhou, C. Z.; Confalonieri, F.; Medina, N.; Zivanovic, Y.; Esnault, C.; Yang, T.; Jacquet, M.; Janin, J.; Duguet, M.; Perasso, R.; Li, Z. G. Fine organization of Bombyx mori fibroin heavy chain gene. Nucleic Acids Res. 2000, 28, 2413−2419. (12) Zhou, C. Z.; Confalonieri, F.; Jacquet, M.; Perasso, R.; Li, Z. G.; Janin, J. Silk fibrin: Structural implications of a remarkable amino acid sequence. Proteins: Struct., Funct., Genet. 2001, 44, 119−122. (13) Kim, H.; Kim, J. S. A guide to genome engineering with programmable nucleases. Nat. Rev. Genet. 2014, 15, 321−334. (14) Takasu, Y.; Sajwan, S.; Daimon, T.; Osanai-Futahashi, M.; Uchino, K.; Sezutsu, H.; Tamura, T.; Zurovec, M. Efficient TALEN construction for Bombyx mori gene targeting. PLoS One 2013, 8, e73458. (15) Nakade, S.; Tsubota, T.; Sakane, Y.; Kume, S.; Sakamoto, N.; Obara, M.; Daimon, T.; Sezutsu, H.; Yamamoto, T.; Sakuma, T.; Suzuki, K. T. Microhomology-mediated end-joining-dependent integration of donor DNA in cells and animals using TALENs and CRISPR/Cas9. Nat. Commun. 2014, 5, 5560. (16) Takasu, Y.; Kobayashi, I.; Tamura, T.; Uchino, K.; Sezutsu, H.; Zurovec, M. Precise genome editing in the silkworm Bombyx mori using TALENs and ds- and ssDNA donors − A practical approach. Insect Biochem. Mol. Biol. 2016, 78, 29−38. (17) Takasu, Y.; Iizuka, T.; Yoshioka, T.; Kameda, T.; Uchino, K.; Sezutsu, H. Genome editing of silk-encoding genes in Bombyx mori. In Conference Abstract in the 5th Asia-Pacific Congress of Sericulture and Insect Biotechnology (APSERI) Bangkok, Thailand, Feb 28−March 2, 2017 ; p 66. (18) Drummy, L. F.; Farmer, B. L.; Naik, R. R. Correlation of the βsheet crystal size in silk fibers with the protein amino acid sequence. Soft Matter 2007, 3, 877−82. (19) Yoshioka, T.; Tashiro, K.; Ohta, N. Molecular orientation enhancement of silk by the hot-stretching-induced transition from αhelix-HFIP complex to β-sheet. Biomacromolecules 2016, 17, 1437− 1488. (20) Lu, Y. H.; Lin, H.; Chen, Y. Y.; Wang, C.; Hua, Y. R. Structure and performance of Bombyx mori silk modified with nano-TiO2 and chitosan. Fibers Polym. 2007, 8, 1−6. (21) Cermak, T.; Doyle, E. L.; Christian, M.; Wang, L.; Zhang, Y.; Schmidt, C.; Baller, J. A.; Somia, N. V.; Bogdanove, A. J.; Voytas, D. F. Efficient design and assembly of custom TALEN and other TAL effector-based constructs for DNA targeting. Nucleic Acids Res. 2011, 39, e82.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsbiomaterials.8b00043. Sequence variations of intervening coding regions; schematic representation of establishing GE mutant strain and SDS-PAGE analysis of G1 cocoon proteins; nucleotide sequence and amino acid sequence of the PCR-amplified region of GE silk; 13C NMR spectra of native and GE silks; stress−strain curves of native and GE silks; and table of mechanical properties for native and GE silks (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: kamedat@affrc.go.jp. ORCID

Taiyo Yoshioka: 0000-0002-5800-312X Tsunenori Kameda: 0000-0001-8456-1857 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Dr. Tetsuya Iizuka (NARO, Japan) for preparing and maintaining the silkworm strains and Dr. Keiro Uchino (NARO, Japan) for microinjection. The synchrotron radiation experiments were performed at the BL40B2 of SPring-8 with the approval of Japan Synchrotron Radiation Research Institute (JASRI) (Proposal 2016A1440). T.Y. and T.K. thank Dr. Noboru OHTA (JASRI, Japan) for his technical support of the synchrotron X-ray experiments at SPring-8 BL40B2.

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REFERENCES

(1) Kameda, T.; Ohkawa, Y.; Yoshizawa, K.; Naito, J.; Ulrich, A. S.; Asakura, T. Hydrogen-Bonding Structure in Serine Side Chains in Bombyx mori and Samia cynthia ricini Silk Fibroin Determined by SolidState 2H NMR. Macromolecules 1999, 32, 7166−7171. (2) Nishikawa, S.; Ono, S. Transmission of X-rays through fibrous, lamellar and granular substances. Proc. Tokyo Math. Phys. Soc. 1913, 7, 131−138. (3) Brill, R. Ü ber Seidenfibroin I. Justus Liebigs Ann. Chem. 1923, 434, 204−217. (4) Warwicker, J. O. The crystal structure of silk fibroin. Acta Crystallogr. 1954, 7, 565−573. (5) Marsh, R. E.; Corey, R. B.; Pauling, L. An investigation of the structure of silk fibroin. Biochim. Biophys. Acta 1955, 16, 1−34. D

DOI: 10.1021/acsbiomaterials.8b00043 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX