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Genetically encoded protein sulfation in mammalian cells

Nature Chemical Biology volume 16, pages 379–382 (2020)Cite this article

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Abstract

Tyrosine sulfation is an important post-translational modification found in higher eukaryotes. Here we report an engineered tyrosyl-tRNA synthetase/tRNA pair that co-translationally incorporates O-sulfotyrosine in response to UAG codons in Escherichia coli and mammalian cells. This platform enables recombinant expression of eukaryotic proteins homogeneously sulfated at chosen sites, which was demonstrated by expressing human heparin cofactor II in mammalian cells in different states of sulfation.

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Fig. 1: Genetically encoding sTyr.
Fig. 2: Expression and biochemical analysis of precisely sulfated HCII.

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Data associated with this work are available upon request from the corresponding author.

References

  1. Moore, K. L. Protein tyrosine sulfation: a critical posttranslation modification in plants and animals. Proc. Natl Acad. Sci. USA 106, 14741–14742 (2009).

    Article  CAS  Google Scholar 

  2. Seibert, C. & Sakmar, T. P. Toward a framework for sulfoproteomics: synthesis and characterization of sulfotyrosine-containing peptides. Biopolymers 90, 459–477 (2008).

    Article  CAS  Google Scholar 

  3. Stone, M. J., Chuang, S., Hou, X., Shoham, M. & Zhu, J. Z. Tyrosine sulfation: an increasingly recognised post-translational modification of secreted proteins. New Biotechnol. 25, 299–317 (2009).

    Article  CAS  Google Scholar 

  4. Yang, Y. S. et al. Tyrosine sulfation as a protein post-translational modification. Molecules (Basel, Switzerland) 20, 2138–2164 (2015).

    Article  Google Scholar 

  5. Farzan, M. et al. Tyrosine sulfation of the amino terminus of CCR5 facilitates HIV-1 entry. Cell 96, 667–676 (1999).

    Article  CAS  Google Scholar 

  6. Huang, C.-c et al. Structural basis of tyrosine sulfation and VH-gene usage in antibodies that recognize the HIV type 1 coreceptor-binding site on gp120. Proc. Natl Acad. Sci. USA 101, 2706–2711 (2004).

    Article  CAS  Google Scholar 

  7. Li, X., Hitomi, J. & Liu, C. C. Characterization of a sulfated anti-HIV antibody using an expanded genetic code. Biochemistry 57, 2903–2907 (2018).

    Article  CAS  Google Scholar 

  8. Stone, M. J. & Payne, R. J. Homogeneous sulfopeptides and sulfoproteins: synthetic approaches and applications to characterize the effects of tyrosine sulfation on biochemical function. Acc. Chem. Res. 48, 2251–2261 (2015).

    Article  CAS  Google Scholar 

  9. Thompson, R. E. et al. Tyrosine sulfation modulates activity of tick-derived thrombin inhibitors. Nat. Chem. 9, 909–917 (2017).

    Article  Google Scholar 

  10. Mikkelsen, J., Thomsen, J. & Ezban, M. Heterogeneity in the tyrosine sulfation Chinese hamster ovary cell produced recombinant FVIII. Biochemistry 30, 1533–1537 (1991).

    Article  CAS  Google Scholar 

  11. Chin, J. W. Expanding and reprogramming the genetic code. Nature 550, 53–60 (2017).

    Article  Google Scholar 

  12. Italia, J. S. et al. Expanding the genetic code of mammalian cells. Biochem. Soc. Trans. 45, 555–562 (2017).

    Article  CAS  Google Scholar 

  13. Young, D. D. & Schultz, P. G. Playing with the molecules of life. ACS Chem. Biol. 13, 854–870 (2018).

    Article  CAS  Google Scholar 

  14. Liu, C. C., Brustad, E., Liu, W. & Schultz, P. G. Crystal structure of a biosynthetic sulfo-hirudin complexed to thrombin. J. Am. Chem. Soc. 129, 10648–10649 (2007).

    Article  CAS  Google Scholar 

  15. Liu, C. C. & Schultz, P. G. Recombinant expression of selectively sulfated proteins in Escherichia coli. Nat. Biotechnol. 24, 1436–1440 (2006).

    Article  CAS  Google Scholar 

  16. Watson, E. E. et al. Mosquito-derived anophelin sulfoproteins are potent antithrombotics. ACS Central Sci. 4, 468–476 (2018).

    Article  CAS  Google Scholar 

  17. Italia, J. S., Latour, C., Wrobel, C. J. & Chatterjee, A. Resurrecting the bacterial tyrosyl-tRNA synthetase/tRNA pair for expanding the genetic code of both E. coli and eukaryotes. Cell Chem. Biol. 25, 1304–1312 (2018).

    Article  CAS  Google Scholar 

  18. Chin, J. W. et al. An expanded eukaryotic genetic code. Science 301, 964–967 (2003).

    Article  CAS  Google Scholar 

  19. Dumas, A., Lercher, L., Spicer, C. D. & Davis, B. G. Designing logical codon reassignment—expanding the chemistry in biology. Chem. Sci. 6, 50–69 (2015).

    Article  CAS  Google Scholar 

  20. Italia, J. S. et al. An orthogonalized platform for genetic code expansion in both bacteria and eukaryotes. Nat. Chem. Biol. 13, 446–450 (2017).

    Article  CAS  Google Scholar 

  21. Tollefsen, D. M. Heparin cofactor II. Adv. Exp. Med. Biol. 425, 35–44 (1997).

    Article  CAS  Google Scholar 

  22. Tollefsen, D. M. Heparin cofactor II modulates the response to vascular injury. Arterioscler. Thromb. Vasc. Biol. 27, 454–460 (2007).

    Article  CAS  Google Scholar 

  23. Hortin, G., Tollefsen, D. & Strauss, A. W. Identification of two sites of sulfation of human heparin cofactor II. J. Biol. Chem. 261, 15827–15830 (1986).

    CAS  PubMed  Google Scholar 

  24. Ciaccia, A. V., Monroe, D. M. & Church, F. C. Arginine 200 of heparin cofactor II promotes intramolecular interactions of the acidic domain implication for thrombin inhibition. J. Biol. Chem. 272, 14074–14079 (1997).

    Article  CAS  Google Scholar 

  25. Mitchell, J. W. & Church, F. C. Aspartic acid residues 72 and 75 and tyrosine-sulfate 73 of heparin cofactor II promote intramolecular interactions during glycosaminoglycan binding and thrombin inhibition. J. Biol. Chem. 277, 19823–19830 (2002).

    Article  CAS  Google Scholar 

  26. Zheng, Y., Lewis, T. L. Jr, Igo, P., Polleux, F. & Chatterjee, A. Virus-enabled optimization and delivery of the genetic machinery for efficient unnatural amino acid mutagenesis in mammalian cells and tissues. ACS Synth. Biol. 6, 13–18 (2016).

    Article  Google Scholar 

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Acknowledgements

We thank D.M. Monroe III (UNC) for helpful discussions on the HCII kinetic assay. This work was supported by the National Institutes of Health (R01GM124319 and R01GM126220 to A.C.; 1R01GM118431 and 1R01GM117004 to E.W.).

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Authors and Affiliations

  1. Department of Chemistry, Boston College, Chestnut Hill, MA, USA

    James S. Italia, Jennifer C. Peeler, Christen M. Hillenbrand, Christopher Latour, Eranthie Weerapana & Abhishek Chatterjee

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  1. James S. Italia
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  2. Jennifer C. Peeler
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  3. Christen M. Hillenbrand
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  4. Christopher Latour
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  6. Abhishek Chatterjee
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Contributions

A.C. and J.S.I. designed the experiments. J.S.I. conducted all experiments. C.M.H. and C.L. assisted with cloning and protein expression. J.C.P. performed the MS analyses of HCII and E.W. supervised. A.C. and J.S.I. prepared the manuscript.

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Correspondence to Abhishek Chatterjee.

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Supplementary Figs. 1–11, Table 1 and Note.

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Italia, J.S., Peeler, J.C., Hillenbrand, C.M. et al. Genetically encoded protein sulfation in mammalian cells. Nat Chem Biol 16, 379–382 (2020). https://doi.org/10.1038/s41589-020-0493-1

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