Articles pubs.acs.org/acschemicalbiology
Backbone Circularization Coupled with Optimization of Connecting Segment in Effectively Improving the Stability of Granulocyte-Colony Stimulating Factor Takamitsu Miyafusa,† Risa Shibuya,‡ Wataru Nishima,† Rie Ohara,‡ Chuya Yoshida,† and Shinya Honda*,†,‡ †
Biomedical Research Institute, The National Institute of Advanced Industrial Science and Technology, Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8566, Japan ‡ Department of Computational Biology and Medical Sciences, Graduate School of Frontier Sciences, The University of Tokyo, 5-1-5 Kashiwanoha, Kashiwa, Chiba 277-8562, Japan S Supporting Information *
ABSTRACT: Backbone circularization of protein is a powerful method to improve its structural stability. In this paper, we presumed that a tight connection leads to much higher stability. Therefore, we designed circularized variants of a granulocytecolony stimulating factor (G-CSF) with a structurally optimized terminal connection. To estimate the appropriate length of the connection, we surveyed the Protein Data Bank to find local structures as a model for the connecting segment. We set the library of local structures composed of “helix−loop−helix,” subsequently selected entries similar to the G-CSF terminus, and finally sorted the hit structures according to the loop length. Two, five, or nine loop residues were frequently observed; thus, three circularized variants (C163, C166, and C170) were constructed, prepared, and evaluated. All circularized variants demonstrated a higher thermal stability than linear G-CSF (L175). In particular, C166 that retained five connecting residues demonstrated apparent Tm values of 69.4 °C, which is 8.7 °C higher than that of the circularized variant with no truncation (C177), indicating that the optimization of the connecting segment is effective for enhancing the overall structural stability. C166 also showed higher proteolytic stability against both endoprotease and exopeptidase than L175. We anticipate that the present study will contribute to the improvement in the general design of circularized protein and development of G-CSF biobetters.
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Assessment for Therapeutic Protein Products.”6 We assumed that circularization of polypeptides is applicable for biopharmaceutical development because it only modifies the terminal portion of the polypeptide and would be less likely to gain immunogenicity. Circularization has succeeded in stabilizing small proteins ( C170 > C177 > C163 > L175) was reflected on the combined effect of circularization and loop shortening. The difference in stability among circularized variants could be explained by the restriction in the entropy.3 Shortening of the linker length restricted the conformational space of the unfolded state and thus shifted the equilibrium toward a folded state. As mentioned in a previous work on the PIN1 WW domain, a linker whose length is either too long or too short had lower stability.38 C166 is the most stable variant among all circularized G-CSFs. The increase in the Tm value by 12.9 °C was extraordinarily high and comparable with that of the most stable point mutant, replacing the 10 residues in the structural core.22 We propose that, in general, circularization with appropriate truncation would be a more promising and safer method than the replacement of many amino acids. The stability against digestion by elastase (C177 ≥ C166 > C170 > C163 = L175) indicated that the proteolytic (and further in vivo) stability is not determined only by the structural stability. We estimated that circularized variants could be stabilized by mainly two mechanisms: high structural stability and tolerance for first digestion to maintain a single linear polypeptide. C177 could be stabilized by some additional effects related to a local amino-acid sequence and/or structure. C170 and C163 meaningfully demonstrated lower stability in both experiments against elastase and carboxypeptidase Y probably because they expose the surface that is readily nonspecifically digestible. Note that we first applied a common method to incubate the proteins in serum at 37 °C and measured the concentration at predetermined time points.39 Unexpectedly, all five variants, including L175, were not digested by the serum incubation even under the condition in which filgrastim was completely digested (data not shown). Compared with filgrastim, the alteration common to all variants was only the substitution of Cys to Ser at position 17 in L175
(Figure 1D). We have no reasonable explanation, but actually, a similar result has been reported from another group.40 Taken together, we succeeded in designing and obtaining a structurally superstable G-CSF by optimizing the connecting segment, where measurement of in vivo stability is required for further investigation. In summary, we have proposed a protein design combined with circularization and loop shortening that is significantly effective for enhancing stability. We emphasized that achieving outstanding optimized values could be realized using only the length. Further optimization by selecting side chains could improve protein stability; however, the scope is beyond our current study. We intend to improve the stability while preserving the original amino-acid sequence because limiting our process to the original sequence can suppress the increase in immunogenicity risk that arises from engineered products. The structure-based approach conducted in the present study provides criteria for determining the length of a connecting segment. The success in producing variants with superior structural and proteolytic stabilities demonstrated the effectiveness of our approach. Yet, further improvement is needed to avoid false candidates such as C163, which consistently showed low stability within all measurements. We could improve the computational method by tuning both the scaffold and library. We used a scaffold structure composed of eight residues (four residues in each of the N- or C-terminal). These values may probably be too small, and thus, the portion might be distorted when applied on the connecting segments of C163. By adopting a larger region in the scaffold, the prediction could become more reliable. However, it raises the possibility of excluding a relatively short helix. Second, our data sets did not exclude structurally homologous proteins, although we omitted the redundant entries based on the primary sequences (similarity ≥90%). Consequently, all 10 structures with a two-residue loop were from the annexin family.41 On the basis of the idea of comparing local structures, we do not need to dispose of the homologues sharing similar overall structure. Nonetheless, eliminating the bias of the population of deposited structures remains important. However, C163 realized an interesting feature that was processed without any linear byproduct. The efficiency of intein circularization is widely accepted to be not very high, and separating the linear byproduct from the circularized main product is challenging because they are chemically and physically similar to each other. This byproduct was produced by the hydrolysis of lactone before peptide bond formation according to the proposed mechanism.9 Shortening the terminal regions could presumably make the reaction site more packed and could block entry of water. All in all, reducing the linear byproduct for the intein technology would be innovative. Hence, the relationship between the connector length and efficiency of circularization should be elucidated in the future. Conclusion. We have designed a circularized G-CSF with a tight and distortion-free connecting segment. By determining an appropriate connector length using the structure-based method, we realized structurally more stable variants than the simply connected one. These results indicate that the combined strategy of circularization and connector optimization is effective. In other words, the engineering of the terminus is critical in enhancing the stability by circularization. We also demonstrated that the circularization efficiency would be dramatically improved in some cases. These observations are E
DOI: 10.1021/acschembio.7b00776 ACS Chem. Biol. XXXX, XXX, XXX−XXX
Articles
ACS Chemical Biology useful for the next challenge of producing ideal circularized protein with high stability and high yield. We also believe that this research will encourage further studies focusing on the improvement of G-CSF, a therapeutic agent that is classically used for cancer therapy and has also been recently proposed for many other diseases such as strokes and Alzheimer’s disease.42,43
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METHODS
Further details of the procedures used in this study are described in the Supporting Information. Expression and Purification of G-CSF Variants. The DNA that encodes L175 and that which encodes split DnaE intein from Nostoc punctiforme were purchased from Takara. DNA fragments encoded for C163, C166, C170, and C177 were amplified from the vector of L175. All the G-CSF variants (L175, C177, C170, C166, and C163) were expressed in E. coli BL21 (DE3) strains and purified from the insoluble fraction. Design of Connecting Segments. The length and amino-acid sequence of the connecting segment were determined by the frequency of appearance in the PDB and free-energy-based (Rosetta score)44 optimization, respectively. Backbone and side-chain optimizations were conducted for all possible sequences of the two-residue connector. We used the FASTER algorithm45 and Rosetta score function44 for the initial construction of the side-chain and rotamer optimizations implemented in the CHOMP program.46 The molecular dynamic package (AMBER)47 and AMBER force field48 were also used for the energy minimization. Digestion by Carboxy Peptidase Y. The G-CSF variants at a concentration of 10 μg/mL were mixed with 100-μg/mL carboxypeptidase Y (Roche) in a buffer containing 100 mM sodium acetate with a pH of 6.5. The solutions were incubated at 37 °C for 1, 2, 3, and 4 h (n = 3 for all samples). Thereafter, the nondegraded G-CSF was quantified by SDS-PAGE. The data were fit to a pseudo-first-order reaction. CD. The CD spectra and CD melting curves were recorded with a J805 spectropolarimeter (JASCO). The proteins were dissolved at 5 μM in 10 mM HEPES-NaOH with a pH of 7.4 and 150 mM NaCl. The spectra were obtained at 10 and 90 °C and presented in units of molecular ellipticity per mole of residue. The melting curves were obtained by monitoring the ellipticity at 222 nm while increasing the temperature from 10 to 90 °C at a heating rate of 1.0 °C/min. SPR. An SPR assay was performed using Biacore T200 (GE Healthcare). Protein A (Nacalai, Japan) was first covalently immobilized on sensor chip CM5 (GE Healthcare) via amine coupling. The human-cell-expressed G-CSF receptor Fc Chimera (SYMANSYS) in a running buffer [10-mM HEPES-NaOH, 150 mm NaCl, 0.05% (v/v) surfactant Tween 20, and pH of 7.4] was then injected and captured by protein A. Kinetic characterization of the GCSF was performed using the single-cycle kinetic method. Sensorgrams were obtained by injecting the G-CSF with increasing concentrations from 1.25 to 20 nM at a flow rate of 30 μL/min. All measurements were performed in duplicate. Digestion by Elastase. The G-CSF variants at a concentration of 10 μg/mL were mixed with 1 μg/mL elastase (Wako) in a buffer containing 100 mM HEPES-NaOH with a pH of 7.4. The solutions were incubated at 37 °C for 15, 30, 45, 60, 90, and 120 min (n = 3 for all samples). The elastase reaction was stopped by adding phenylmethanesulfonyl fluoride at a final concentration of 1 mM. Thereafter, the nondegraded G-CSF was quantified using the AlphaLISA technique, EnVision-Alpha Reader (PerkinElmer), and Human GCSF Kit (PerkinElmer) according to the manufacturer’s instructions.
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Chromatograms of the purification using a MonoQ column, deconvoluted mass spectrum of C177, SPR sensorgrams to measure the affinity of circularized GCSF to G-CSF receptor, prediction of immunogenicity, number of loop templates extracted from the PDB, top 10 optimal sequences, and methods including DNA sequences used for cloning (PDF)
AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. ORCID
Shinya Honda: 0000-0002-7878-3944 Notes
The authors declare the following competing financial interest(s): We declare that two of the authors (T.M. and S.H.) are inventors with a pending patent for the developed molecules.
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ACKNOWLEDGMENTS We would like to thank S. Yoshida for his contribution in preparing the expression vectors and establishing the preparation method. This work was supported in part by grants from the Japan Society for the Promotion of Science [Grant Number 23510273 (to S.H.)].
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REFERENCES
(1) Kang, H. J., and Baker, E. N. (2009) Intramolecular isopeptide bonds give thermodynamic and proteolytic stability to the major Pilin protein of Streptococcus pyogenes. J. Biol. Chem. 284, 20729−20737. (2) Kanaya, S., Katsuda, C., Kimura, S., Nakai, T., Kitakani, E., Nakamura, H., Katayanagi, K., Morikawa, K., and Ikehara, M. (1991) Stabilization of Escherichia coli ribonuclease H by introduction of an artificial disulfide bond. J. Biol. Chem. 266, 6038−6044. (3) Zhou, H. X. (2004) Loops, Linkages, Rings, Catenanes, Cages, and Crowders: Entropy-Based Strategies for Stabilizing Proteins. Acc. Chem. Res. 37, 123−130. (4) Dagan, S., Hagai, T., Gavrilov, Y., Kapon, R., Levy, Y., and Reich, Z. (2013) Stabilization of a protein conferred by an increase in folded state entropy. Proc. Natl. Acad. Sci. U. S. A. 110, 10628−10633. (5) Gavrilov, Y., Dagan, S., and Levy, Y. (2015) Shortening a loop can increase protein native state entropy. Proteins: Struct., Funct., Genet. 83, 2137−2146. (6) (2014) Guidance for Industry Immunogenicity Assessment for Therapeutic Protein Products, Food and Drug Administration, Washington, DC. (7) Camarero, J. A., Fushman, D., Sato, S., Giriat, I., Cowburn, D., Raleigh, D. P., and Muir, T. W. (2001) Rescuing a destabilized protein fold through backbone cyclization. J. Mol. Biol. 308, 1045−1062. (8) Aboye, T. L., and Camarero, J. A. (2012) Biological synthesis of circular polypeptides. J. Biol. Chem. 287, 27026−27032. (9) Scott, C. P., Abel-Santos, E., Wall, M., Wahnon, D. C., and Benkovic, S. J. (1999) Production of cyclic peptides and proteins in vivo. Proc. Natl. Acad. Sci. U. S. A. 96, 13638−13643. (10) Parthasarathy, R., Subramanian, S., and Boder, E. T. (2007) Sortase A as a novel molecular “stapler” for sequence-specific protein conjugation. Bioconjugate Chem. 18, 469−476. (11) Chevalier, B. S. (2001) Homing endonucleases: structural and functional insight into the catalysts of intron/intein mobility. Nucleic Acids Res. 29, 3757−3774. (12) Paulus, H. (2000) Protein splicing and related forms of protein autoprocessing. Annu. Rev. Biochem. 69, 447−496. (13) Iwai, H., and Plückthun, A. (1999) Circular β-lactamase: stability enhancement by cyclizing the backbone. FEBS Lett. 459, 166− 172.
ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acschembio.7b00776. F
DOI: 10.1021/acschembio.7b00776 ACS Chem. Biol. XXXX, XXX, XXX−XXX
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ACS Chemical Biology (14) Iwai, H., Lingel, A., and Pluckthun, A. (2001) Cyclic green fluorescent protein produced in vivo using an artificially split PI-PfuI intein from Pyrococcus furiosus. J. Biol. Chem. 276, 16548−16554. (15) Tavassoli, A., and Benkovic, S. J. (2007) Split-intein mediated circular ligation used in the synthesis of cyclic peptide libraries in E. coli. Nat. Protoc. 2, 1126−1133. (16) Hill, C. P., Osslund, T. D., and Eisenberg, D. (1993) The structure of granulocyte-colony-stimulating factor and its relationship to other growth factors. Proc. Natl. Acad. Sci. U. S. A. 90, 5167−5171. (17) Kuga, T., Komatsu, Y., Yamasaki, M., Sekine, S., Miyaji, H., Nishi, T., Sato, M., Yokoo, Y., Asano, M., Okabe, M., Morimoto, M., and Itoh, S. (1989) Mutagenesis of human granulocyte colony stimulating factor. Biochem. Biophys. Res. Commun. 159, 103−111. (18) Frampton, J. E., Lee, C. R., and Faulds, D. (1994) Filgrastim. Drugs 48, 731−760. (19) Walsh, G. (2014) Biopharmaceutical benchmarks 2014. Nat. Biotechnol. 32, 992−1000. (20) Sarkar, C. A., Lowenhaupt, K., Horan, T., Boone, T. C., Tidor, B., and Lauffenburger, D. A. (2002) Rational cytokine design for increased lifetime and enhanced potency using pH-activated “histidine switching. Nat. Biotechnol. 20, 908−913. (21) Arakawa, T., Prestrelski, S. J., Narhi, L. O., Boone, T. C., and Kenney, W. C. (1993) Cysteine 17 of recombinant human granulocyte-colony stimulating factor is partially solvent-exposed. J. Protein Chem. 12, 525−531. (22) Luo, P., Hayes, R. J., Chan, C., Stark, D. M., Hwang, M. Y., Jacinto, J. M., Juvvadi, P., Chung, H. S., Kundu, A., Ary, M. L., and Dahiyat, B. I. (2002) Development of a cytokine analog with enhanced stability using computational ultrahigh throughput screening. Protein Sci. 11, 1218−1226. (23) Piedmonte, D. M., and Treuheit, M. J. (2008) Formulation of Neulasta (pegfilgrastim). Adv. Drug Delivery Rev. 60, 50−58. (24) Popp, M. W., Dougan, S. K., Chuang, T.-Y., Spooner, E., and Ploegh, H. L. (2011) Sortase-catalyzed transformations that improve the properties of cytokines. Proc. Natl. Acad. Sci. U. S. A. 108, 3169− 3174. (25) Tamada, T., Honjo, E., Maeda, Y., Okamoto, T., Ishibashi, M., Tokunaga, M., and Kuroki, R. (2006) Homodimeric cross-over structure of the human granulocyte colony-stimulating factor (GCSF) receptor signaling complex. Proc. Natl. Acad. Sci. U. S. A. 103, 3135−3140. (26) Zheng, W., Olson, J., Vakharia, V., and Tao, Y. J. (2013) The Crystal Structure and RNA-Binding of an Orthomyxovirus Nucleoprotein. PLoS Pathog. 9, e1003624. (27) Tai, M. S., Mudgett-Hunter, M., Levinson, D., Wu, G. M., Haber, E., Oppermann, H., and Huston, J. S. (1990) A bifunctional fusion protein containing Fc-binding fragment B of staphylococcal protein A amino-terminal to antidigoxin single-chain Fv. Biochemistry 29, 8024−8030. (28) Iwai, H., Züger, S., Jin, J., and Tam, P.-H. (2006) Highly efficient protein trans-splicing by a naturally split DnaE intein from Nostoc punctiforme. FEBS Lett. 580, 1853−1858. (29) van Lieshout, J. F. T., Pérez Gutiérrez, O. N., Vroom, W., Planas, A., de Vos, W. M., van der Oost, J., and Koutsopoulos, S. (2012) Thermal stabilization of an endoglucanase by cyclization. Appl. Biochem. Biotechnol. 167, 2039−2053. (30) Jung, G., Ueno, H., and Hayashi, R. (1999) Carboxypeptidase Y: structural basis for protein sorting and catalytic triad. J. Biochem. 126, 1−6. (31) Kolvenbach, C. G., Narhi, L. O., Philo, J. S., Li, T., Zhang, M., and Arakawa, T. (1997) Granulocyte-colony stimulating factor maintains a thermally stable, compact, partially folded structure at pH2. J. Pept. Res. 50, 310−318. (32) Natalello, A., Ami, D., Collini, M., D’Alfonso, L., Chirico, G., Tonon, G., Scaramuzza, S., Schrepfer, R., and Doglia, S. M. (2012) Biophysical characterization of Met-G-CSF: Effects of different sitespecific mono-pegylations on protein stability and aggregation. PLoS One 7, e42511.
(33) Hunter, M. G., Druhan, L. J., Massullo, P. R., and Avalos, B. R. (2003) Proteolytic Cleavage of Granulocyte Colony-Stimulating Factor and its Receptor by Neutrophil Elastase Induces Growth Inhibition and Decreased Cell Surface Expression of the Granulocyte Colony-Stimulating Factor Receptor. Am. J. Hematol. 74, 149−155. (34) El Ouriaghli, F., Fujiwara, H., Metenhorst, J. J., Sconocchia, G., Hensel, N., and Barrett, A. J. (2003) Neutrophil elastase enzymatically antagonizes the in vitro action of G-CSF: Implications for the regulation of granulopoiesis. Blood 101, 1752−1758. (35) Zhang, L., Chen, Y., Wong, H. S., Zhou, S., Mamitsuka, H., and Zhu, S. (2012) Tepitopepan: Extending tepitope for peptide binding prediction covering over 700 hla-dr molecules. PLoS One 7, e30483. (36) Sturniolo, T., Bono, E., Ding, J., Raddrizzani, L., Tuereci, O., Sahin, U., Braxenthaler, M., Gallazzi, F., Protti, M. P., Sinigaglia, F., and Hammer, J. (1999) Generation of tissue-specific and promiscuous HLA ligand databases using DNA microarrays and virtual HLA class II matrices. Nat. Biotechnol. 17, 555−561. (37) Southwood, S., Sidney, J., Kondo, A., del Guercio, M. F., Appella, E., Hoffman, S., Kubo, R. T., Chesnut, R. W., Grey, H. M., and Sette, A. (1998) Several common HLA-DR types share largely overlapping peptide binding repertoires. J. Immunol. 160, 3363−3373. (38) Deechongkit, S., and Kelly, J. W. (2002) The effect of backbone cyclization on the thermodynamics of beta-sheet unfolding: Stability optimization of the PIN WW domain. J. Am. Chem. Soc. 124, 4980− 4986. (39) Stone, E., Chantranupong, L., Gonzalez, C., O’Neal, J., Rani, M., VanDenBerg, C., and Georgiou, G. (2012) Strategies for optimizing the serum persistence of engineered human arginase I for cancer therapy. J. Controlled Release 158, 171−179. (40) Carter, C. R. D., Keeble, J. R., and Thorpe, R. (2004) Human serum inactivates non-glycosylated but not glycosylated granulocyte colony stimulating factor by a protease dependent mechanism: Significance of carbohydrates on the glycosylated molecule. Biologicals 32, 37−47. (41) Gerke, V., and Moss, S. E. (2002) Annexins: from structure to function. Physiol. Rev. 82, 331−371. (42) Schabitz, W.-R., Kollmar, R., Schwaninger, M., Juettler, E., Bardutzky, J., Scholzke, M. N., Sommer, C., and Schwab, S. (2003) Neuroprotective Effect of Granulocyte Colony-Stimulating Factor After Focal Cerebral Ischemia. Stroke 34, 745−751. (43) Tsai, K.-J., Tsai, Y.-C., and Shen, C.-K. J. (2007) G-CSF rescues the memory impairment of animal models of Alzheimer’s disease. J. Exp. Med. 204, 1273−1280. (44) Rohl, C. A., Strauss, C. E. M., Misura, K. M. S., and Baker, D. (2004) Protein structure prediction using Rosetta. Methods Enzymol. 383, 66−93. (45) Allen, B. D., and Mayo, S. L. (2006) Dramatic performance enhancements for the FASTER optimization algorithm. J. Comput. Chem. 27, 1071−1075. (46) Loksha, I. V., Maiolo, J. R., Hong, C. W., Ng, A., and Snow, C. D. (2009) SHARPEN-systematic hierarchical algorithms for rotamers and proteins on an extended network. J. Comput. Chem. 30, 999−1005. (47) Case, D. A., Cheatham, T. E., Darden, T., Gohlke, H., Luo, R., Merz, K. M., Onufriev, A., Simmerling, C., Wang, B., and Woods, R. J. (2005) The Amber biomolecular simulation programs. J. Comput. Chem. 26, 1668−1688. (48) Ponder, J. W., and Case, D. A. (2003) Force fields for protein simulations. Adv. Protein Chem. 66, 27−85.
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DOI: 10.1021/acschembio.7b00776 ACS Chem. Biol. XXXX, XXX, XXX−XXX
