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Bioactive site-specifically modified proteins for 4D patterning of gel biomaterials

Abstract

Protein-modified biomaterials can be used to modulate cellular function in three dimensions. However, as the dynamic heterogeneous control over complex cell physiology continues to be sought, strategies that permit a reversible and user-defined tethering of fragile proteins to materials remain in great need. Here we introduce a modular and robust semisynthetic approach to reversibly pattern cell-laden hydrogels with site-specifically modified proteins. Exploiting a versatile sortase-mediated transpeptidation, we generate a diverse library of homogeneous, singly functionalized proteins with bioorthogonal reactive handles for biomaterial modification. We demonstrate the photoreversible immobilization of fluorescent proteins, enzymes and growth factors to gels with excellent spatiotemporal resolution while retaining native protein bioactivity. Localized epidermal growth factor presentation enables dynamic regulation over proliferation, intracellular mitogen-activated protein kinase signalling and subcellularly resolved receptor endocytosis. Our method broadly permits the modification and patterning of a wide range of proteins, which provides newfound avenues to probe and direct advanced cellular fates in four dimensions.

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Fig. 1: Generation of sortagged protein library for biomaterial modification.
Fig. 2: Comparing the activity of differently modified proteins.
Fig. 3: Photopatterned alteration of hydrogel biomaterials with sortagged proteins.
Fig. 4: 4D photoevolution of hydrogel biomaterials patterned with multiple sortagged proteins.
Fig. 5: Spatial patterning of gels with bioactive site-specifically modified enzymes and growth factors.
Fig. 6: Modulating cell fate with a photoreleasable sortagged fluorophore growth factor chimeric protein.

Data availability

Plasmids and data that support the findings of this study are available from the corresponding author upon reasonable request.

References

  1. Lutolf, M. P., Gilbert, P. M. & Blau, H. M. Designing materials to direct stem-cell fate. Nature 462, 433–441 (2009).

    Article  CAS  Google Scholar 

  2. Tibbitt, M. W. & Anseth, K. S. Dynamic microenvironments: the fourth dimension. Sci. Transl. Med. 4, 160ps24 (2012).

    Article  Google Scholar 

  3. Baker, B. M. & Chen, C. S. Deconstructing the third dimension—how 3D culture microenvironments alter cellular cues. J. Cell Sci. 125, 3015–3024 (2012).

    Article  CAS  Google Scholar 

  4. DeForest, C. A. & Anseth, K. S. Advances in bioactive hydrogels to probe and direct cell fate. Annu. Rev. Chem. Biomol. Eng. 3, 421–444 (2012).

    Article  CAS  Google Scholar 

  5. Burdick, J. A. & Murphy, W. L. Moving from static to dynamic complexity in hydrogel design. Nat. Commun. 3, 1269 (2012).

    Article  Google Scholar 

  6. Langer, R. & Vacanti, J. P. Tissue engineering. Science 260, 920–926 (1993).

    Article  CAS  Google Scholar 

  7. Lee, K. Y. & Mooney, D. J. Hydrogels for tissue engineering. Chem. Rev. 101, 1869–1879 (2001).

    Article  CAS  Google Scholar 

  8. Langer, R. & Tirrell, D. A. Designing materials for biology and medicine. Nature 428, 487–492 (2004).

    Article  CAS  Google Scholar 

  9. Cushing, M. C. & Anseth, K. S. Hydrogel cell cultures. Science 316, 1133–1134 (2007).

    Article  CAS  Google Scholar 

  10. Tibbitt, M. W. & Anseth, K. S. Hydrogels as extracellular matrix mimics for 3D cell culture. Biotechnol. Bioeng. 103, 655–663 (2009).

    Article  CAS  Google Scholar 

  11. Seliktar, D. Designing cell-compatible hydrogels for biomedical applications. Science 336, 1124–1128 (2012).

    Article  CAS  Google Scholar 

  12. Lutolf, M. P. & Hubbell, J. A. Synthetic biomaterials as instructive extracellular microenvironments for morphogenesis in tissue engineering. Nat. Biotechnol. 23, 47–55 (2005).

    Article  CAS  Google Scholar 

  13. Zhang, Y. S., . & Khademhosseini, A. Advances in engineering hydrogels. Science. 356, eaaf362 (2017).

    Article  Google Scholar 

  14. Tam, R. Y., Smith, L. J. & Shoichet, M. S. Engineering cellular microenvironments with photo- and enzymatically responsive hydrogels: toward biomimetic 3D cell culture models. Acc. Chem. Res. 50, 703–713 (2017).

    Article  CAS  Google Scholar 

  15. Caliari, S. R. & Burdick, J. A. A practical guide to hydrogels for cell culture. Nat. Methods 13, 405–414 (2016).

    Article  CAS  Google Scholar 

  16. Rosales, A. M. & Anseth, K. S. The design of reversible hydrogels to capture extracellular matrix dynamics. Nat. Rev. Mater. 1, 15012 (2016).

    Article  CAS  Google Scholar 

  17. Ruskowitz, E. R. & DeForest, C. A. Photoresponsive biomaterials for targeted drug delivery and 4D cell culture. Nat. Rev. Mater. 3, 17087 (2018).

    Article  CAS  Google Scholar 

  18. Luo, Y. & Shoichet, M. S. A photolabile hydrogel for guided three-dimensional cell growth and migration. Nat. Mater. 3, 249–253 (2004).

    Article  CAS  Google Scholar 

  19. Hahn, M. S., Miller, J. S. & West, J. L. Three-dimensional biochemical and biomechanical patterning of hydrogels for guiding cell behavior. Adv. Mater. 18, 2679–2684 (2006).

    Article  CAS  Google Scholar 

  20. DeForest, C. A., Polizzotti, B. D. & Anseth, K. S. Sequential click reactions for synthesizing and patterning three-dimensional cell microenvironments. Nat. Mater. 8, 659–664 (2009).

    Article  CAS  Google Scholar 

  21. DeForest, C. A., Sims, E. A. & Anseth, K. S. Peptide-functionalized click hydrogels with independently tunable mechanics and chemical functionality for 3D cell culture. Chem. Mater. 22, 4783–4790 (2010).

    Article  CAS  Google Scholar 

  22. DeForest, C. A. & Anseth, K. S. Cytocompatible click-based hydrogels with dynamically tunable properties through orthogonal photoconjugation and photocleavage reactions. Nat. Chem. 3, 925–931 (2011).

    Article  CAS  Google Scholar 

  23. DeForest, C. A. & Anseth, K. S. Photoreversible patterning of biomolecules within click-based hydrogels. Angew. Chem. Int. Ed. 51, 1816–1819 (2012).

    Article  CAS  Google Scholar 

  24. Wylie, R. G. et al. Spatially controlled simultaneous patterning of multiple growth factors in three-dimensional hydrogels. Nat. Mater. 10, 799–806 (2011).

    Article  CAS  Google Scholar 

  25. Mosiewicz, K. A. et al. In situ cell manipulation through enzymatic hydrogel photopatterning. Nat. Mater. 12, 1071–1077 (2013).

    Article  Google Scholar 

  26. Griffin, D. R. et al. Hybrid photopatterned enzymatic reaction (HyPER) for in situ cell manipulation. ChemBioChem 15, 233–242 (2014).

    Article  CAS  Google Scholar 

  27. DeForest, C. A. & Tirrell, D. A. A photoreversible protein-patterning approach for guiding stem cell fate in three-dimensional gels. Nat. Mater. 14, 523–531 (2015).

    Article  CAS  Google Scholar 

  28. Fisher, S. A., Baker, A. E. G. & Shoichet, M. S. Designing peptide and protein modified hydrogels: selecting the optimal conjugation strategy. J. Am. Chem. Soc. 139, 7416–7427 (2017).

    Article  CAS  Google Scholar 

  29. Baslé, E., Joubert, N. & Pucheault, M. Protein chemical modification on endogenous amino acids. Chem. Biol. 17, 213–227 (2010).

    Article  Google Scholar 

  30. Rabuka, D. Chemoenzymatic methods for site-specific protein modification. Curr. Opin. Chem. Biol. 14, 790–796 (2010).

    Article  CAS  Google Scholar 

  31. Rabuka, D., Rush, J. S., deHart, G. W., Wu, P. & Bertozzi, C. R. Site-specific chemical protein conjugation using genetically encoded aldehyde tags. Nat. Protoc. 7, 1052–1067 (2012).

    Article  CAS  Google Scholar 

  32. Kulkarni, C., Kinzer-Ursem, T. L. & Tirrell, D. A. Selective functionalization of the protein N terminus with N-myristoyl transferase for bioconjugation in cell lysate. ChemBioChem 14, 1958–1962 (2013).

    Article  CAS  Google Scholar 

  33. Chen, I., Howarth, M., Lin, W. & Ting, A. Y. Site-specific labeling of cell surface proteins with biophysical probes using biotin ligase. Nat. Methods 2, 99–104 (2005).

    Article  CAS  Google Scholar 

  34. Guimaraes, C. P. et al. Site-specific C-terminal and internal loop labeling of proteins using sortase-mediated reactions. Nat. Protoc. 8, 1787–1799 (2013).

    Article  Google Scholar 

  35. Mao, H., Hart, S. A., Schink, A. & Pollok, B. A. Sortase-mediated protein ligation: a new method for protein engineering. J. Am. Chem. Soc. 126, 2670–2671 (2004).

    Article  CAS  Google Scholar 

  36. Mazmanian, S. K., Liu, G., Hung, T. T. & Schneewind, O. Staphylococcus aureus sortase, an enzyme that anchors surface proteins to the cell wall. Science 285, 760–763 (1999).

    Article  CAS  Google Scholar 

  37. Warden-Rothman, R., Caturegli, I., Popik, V. & Tsourkas, A. Sortase-tag expressed protein ligation: combining protein purification and site-specific bioconjugation into a single step. Anal. Chem. 85, 11090–11097 (2013).

    Article  CAS  Google Scholar 

  38. Komatsu, N. et al. Development of an optimized backbone of FRET biosensors for kinases and GTPases. Mol. Biol. Cell 22, 4647–4656 (2011).

    Article  CAS  Google Scholar 

  39. Hermanson, G. T. Bioconjugate Techniques (Academic, Cambridge, 2013).

  40. Farahani, P. E., Adelmund, S. M., Shadish, J. A. & DeForest, C. A. Photomediated oxime ligation as a bioorthogonal tool for spatiotemporally-controlled hydrogel formation and modification. J. Mater. Chem. B 5, 4435–4442 (2017).

    Article  CAS  Google Scholar 

  41. Kloxin, A. M., Kasko, A. M., Salinas, C. N. & Anseth, K. S. Photodegradable hydrogels for dynamic tuning of physical and chemical properties. Science 324, 59–63 (2009).

    Article  CAS  Google Scholar 

  42. Bieniarz, C., Young, D. F. & Cornwell, M. J. Chromogenic redox assay for β-lactamases yielding water-insoluble products. I. Kinetic behavior and redox chemistry. Anal. Biochem. 207, 321–328 (1992).

    Article  CAS  Google Scholar 

  43. Kuhl, P. R. & Griffith-Cima, L. G. Tethered epidermal growth factor as a paradigm for growth factor-induced stimulation from the solid phase. Nat. Med. 2, 1022–1027 (1996).

    Article  CAS  Google Scholar 

  44. Fan, V. H. et al. Tethered epidermal growth factor provides a survival advantage to mesenchymal stem cells. Stem Cells 25, 1241–1251 (2007).

    Article  CAS  Google Scholar 

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Acknowledgements

The authors recognize and thank R. Seifert and D. Hailey of the University of Washington Garvey Imaging Center for their ongoing support and advice, F. Watt and T. Hiratsuka (King’s College London) for helpful discussion on visualizing the MAPK activation, M. Matsuda (Kyoto University) for a gift of the HeLa cells transfected with the EKAREV FRET sensor, as well as R. Warden-Rothman and A. Tsourkis (University of Pennsylvania) for providing the pSTEPL plasmid37. The authors thank B. Badeau for assistance in synthesizing N3-oNB-OSu, S. Adelmund for providing BCN-OSu and A. Im for help with the protein purification optimization. We acknowledge support from S. Edgar at the UW Mass Spectrometry Center as well as that from the NIH and N. Peters at the UW W. M. Keck Microscopy Center (S10 OD016240). This work was supported by a University of Washington Faculty Startup Grant (C.A.D.), a Jaconette L. Tietze Young Scientist Research Award (C.A.D.) and a CAREER Award (DMR 1652141, C.A.D.) from the National Science Foundation.

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Contributions

For this manuscript, J.A.S. and C.A.D. conceived and designed the experiments; J.A.S. and G.M.B. performed the experiments; J.A.S. and C.A.D. analysed the data and prepared the figures; J.A.S. and C.A.D. wrote the paper.

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Correspondence to Cole A. DeForest.

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Supplementary information

Supplementary Information

Supplementary Figs. 1–23, Supplementary Table 1, Supplementary Methods, Supplementary Video Captions 1–3, Supplementary References 1–5.

Reporting Summary

Supplementary Video 1

Scanned xy planes of multiphoton ‘cell’ pattern from Fig. 4f–i rendered using Imaris.

Supplementary Video 2

Scanned xz planes of multiphoton ‘cell’ pattern from Figure 4f–i rendered using Imaris.

Supplementary Video 3

Time-lapse FRET quantification of HeLa cells expressing EKAREV.

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Shadish, J.A., Benuska, G.M. & DeForest, C.A. Bioactive site-specifically modified proteins for 4D patterning of gel biomaterials. Nat. Mater. 18, 1005–1014 (2019). https://doi.org/10.1038/s41563-019-0367-7

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