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Redirecting lipoic acid ligase for cell surface protein labeling with small-molecule probes

Nature Biotechnology volume 25, pages 1483–1487 (2007)Cite this article

Abstract

Live cell imaging is a powerful method to study protein dynamics at the cell surface, but conventional imaging probes are bulky, or interfere with protein function1,2, or dissociate from proteins after internalization3,4. Here, we report technology for covalent, specific tagging of cellular proteins with chemical probes. Through rational design, we redirected a microbial lipoic acid ligase (LplA)5 to specifically attach an alkyl azide onto an engineered LplA acceptor peptide (LAP). The alkyl azide was then selectively derivatized with cyclo-octyne6 conjugates to various probes. We labeled LAP fusion proteins expressed in living mammalian cells with Cy3, Alexa Fluor 568 and biotin. We also combined LplA labeling with our previous biotin ligase labeling7,8, to simultaneously image the dynamics of two different receptors, coexpressed in the same cell. Our methodology should provide general access to biochemical and imaging studies of cell surface proteins, using small fluorophores introduced via a short peptide tag.

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Figure 1: Redirecting LplA for site-specific protein labeling with fluorescent probes.
Figure 2: LplA labels the LAP peptide without modifying endogenous mammalian proteins.
Figure 3: Site-specific labeling of LAP fusion proteins with fluorophores.
Figure 4: Simultaneous labeling and imaging of two receptors in polarized cells in a wound healing assay.

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References

  1. Debant, A., Ponzio, G., Clauser, E., Contreres, J.O. & Rossi, B. Receptor cross-linking restores an insulin metabolic effect altered by mutation on tyrosine 1162 and tyrosine 1163. Biochemistry 28, 14–17 (1989).

    Article  CAS  Google Scholar 

  2. Weiss, A. & Littman, D.R. Signal transduction by lymphocyte antigen receptors. Cell 76, 263–274 (1994).

    Article  CAS  Google Scholar 

  3. Anderson, R.G., Brown, M.S., Beisiegel, U. & Goldstein, J.L. Surface distribution and recycling of the low density lipoprotein receptor as visualized with antireceptor antibodies. J. Cell Biol. 93, 523–531 (1982).

    Article  CAS  Google Scholar 

  4. Barak, L.S. & Webb, W.W. Fluorescent low density lipoprotein for observation of dynamics of individual receptor complexes on cultured human fibroblasts. J. Cell Biol. 90, 595–604 (1981).

    Article  CAS  Google Scholar 

  5. Green, D.E., Morris, T.W., Green, J., Cronan, J.E., Jr. & Guest, J.R. Purification and properties of the lipoate protein ligase of Escherichia coli. Biochem. J. 309, 853–862 (1995).

    Article  CAS  Google Scholar 

  6. Agard, N.J., Baskin, J.M., Prescher, J.A., Lo, A. & Bertozzi, C.R. A comparative study of bioorthogonal reactions with azides. ACS Chem. Biol. 1, 644–648 (2006).

    Article  CAS  Google Scholar 

  7. Howarth, M., Takao, K., Hayashi, Y. & Ting, A.Y. Targeting quantum dots to surface proteins in living cells with biotin ligase. Proc. Natl. Acad. Sci. USA 102, 7583–7588 (2005).

    Article  CAS  Google Scholar 

  8. Howarth, M. et al. A monovalent streptavidin with a single femtomolar biotin binding site. Nat. Methods 3, 267–273 (2006).

    Article  CAS  Google Scholar 

  9. Marks, K.M. & Nolan, G.P. Chemical labeling strategies for cell biology. Nat. Methods 3, 591–596 (2006).

    Article  CAS  Google Scholar 

  10. Prescher, J.A. & Bertozzi, C.R. Chemistry in living systems. Nat. Chem. Biol. 1, 13–21 (2005).

    Article  CAS  Google Scholar 

  11. Ali, S.T. & Guest, J.R. Isolation and characterization of lipoylated and unlipoylated domains of the E2p subunit of the pyruvate dehydrogenase complex of Escherichia coli. Biochem. J. 271, 139–145 (1990).

    Article  CAS  Google Scholar 

  12. Fujiwara, K. et al. Crystal structure of lipoate-protein ligase A from Escherichia coli. Determination of the lipoic acid-binding site. J. Biol. Chem. 280, 33645–33651 (2005).

    Article  CAS  Google Scholar 

  13. Green, J.D., Laue, E.D., Perham, R.N., Ali, S.T. & Guest, J.R. Three-dimensional structure of a lipoyl domain from the dihydrolipoyl acetyltransferase component of the pyruvate dehydrogenase multienzyme complex of Escherichia coli. J. Mol. Biol. 248, 328–343 (1995).

    CAS  PubMed  Google Scholar 

  14. Kiick, K.L., Saxon, E., Tirrell, D.A. & Bertozzi, C.R. Incorporation of azides into recombinant proteins for chemoselective modification by the Staudinger ligation. Proc. Natl. Acad. Sci. USA 99, 19–24 (2002).

    Article  CAS  Google Scholar 

  15. Griffin, R.J. The medicinal chemistry of the azido group. Prog. Med. Chem. 31, 121–232 (1994).

    Article  CAS  Google Scholar 

  16. 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 

  17. Lin, C.W. & Ting, A.Y. Transglutaminase-catalyzed site-specific conjugation of small-molecule probes to proteins in vitro and on the surface of living cells. J. Am. Chem. Soc. 128, 4542–4543 (2006).

    Article  CAS  Google Scholar 

  18. Adams, S.R. et al. New biarsenical ligands and tetracysteine motifs for protein labeling in vitro and in vivo: synthesis and biological applications. J. Am. Chem. Soc. 124, 6063–6076 (2002).

    Article  CAS  Google Scholar 

  19. Willnow, T.E. The low-density lipoprotein receptor gene family: multiple roles in lipid metabolism. J. Mol. Med. 77, 306–315 (1999).

    Article  CAS  Google Scholar 

  20. Reche, P. & Perham, R.N. Structure and selectivity in post-translational modification: attaching the biotinyl-lysine and lipoyl-lysine swinging arms in multifunctional enzymes. EMBO J. 18, 2673–2682 (1999).

    Article  CAS  Google Scholar 

  21. Pasquale, E.B. Eph receptor signalling casts a wide net on cell behaviour. Nat. Rev. Mol. Cell Biol. 6, 462–475 (2005).

    Article  CAS  Google Scholar 

  22. Singh, A.B. & Harris, R.C. Autocrine, paracrine and juxtacrine signaling by EGFR ligands. Cell. Signal. 17, 1183–1193 (2005).

    Article  CAS  Google Scholar 

  23. Tuli, S.S. et al. Immunohistochemical localization of EGF, TGF-alpha, TGF-beta, and their receptors in rat corneas during healing of excimer laser ablation. Curr. Eye Res. 31, 709–719 (2006).

    Article  CAS  Google Scholar 

  24. Flanagan, J.G. & Vanderhaeghen, P. The ephrins and Eph receptors in neural development. Annu. Rev. Neurosci. 21, 309–345 (1998).

    Article  CAS  Google Scholar 

  25. Wimmer-Kleikamp, S.H. & Lackmann, M. Eph-modulated cell morphology, adhesion and motility in carcinogenesis. IUBMB Life 57, 421–431 (2005).

    Article  CAS  Google Scholar 

  26. George, N., Pick, H., Vogel, H., Johnsson, N. & Johnsson, K. Specific labeling of cell surface proteins with chemically diverse compounds. J. Am. Chem. Soc. 126, 8896–8897 (2004).

    Article  CAS  Google Scholar 

  27. Griffin, B.A., Adams, S.R. & Tsien, R.Y. Specific covalent labeling of recombinant protein molecules inside live cells. Science 281, 269–272 (1998).

    Article  CAS  Google Scholar 

  28. Brock, R., Hamelers, I.H. & Jovin, T.M. Comparison of fixation protocols for adherent cultured cells applied to a GFP fusion protein of the epidermal growth factor receptor. Cytometry 35, 353–362 (1999).

    Article  CAS  Google Scholar 

  29. McLean, A.J. & Milligan, G. Ligand regulation of green fluorescent protein-tagged forms of the human beta(1)- and beta(2)-adrenoceptors; comparisons with the unmodified receptors. Br. J. Pharmacol. 130, 1825–1832 (2000).

    Article  CAS  Google Scholar 

  30. Zhou, Z. et al. Genetically encoded short peptide tags for orthogonal protein labeling by Sfp and AcpS phosphopantetheinyl transferases. ACS Chem. Biol. 2, 337–346 (2007).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

The authors thank Mark Howarth, John Cronan, Irwin Chen, Chi-Wang Lin, Robin Prince and Martin Lackmann for their assistance and advice. This work was supported by the National Institutes of Health (R01 GM072670-01 to A.Y.T. and GM58867 to C.R.B.), the Sloan Foundation, the Dreyfus Foundation, a La Caixa Foundation predoctoral fellowship (to M.F.-S.), and National Science Foundation and National Science Defense and Engineering predoctoral fellowships (to J.M.B.).

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Authors and Affiliations

  1. Department of Chemistry, Massachusetts Institute of Technology, 77 Massachusetts Ave., Cambridge, 02139, Massachusetts, USA

    Marta Fernández-Suárez, Hemanta Baruah, Laura Martínez-Hernández, Kathleen T Xie & Alice Y Ting

  2. Department of Chemistry, University of California, Berkeley, 94720, California, USA

    Jeremy M Baskin & Carolyn R Bertozzi

  3. Department of Molecular and Cell Biology, University of California, Berkeley, California 94720, USA.,

    Carolyn R Bertozzi

  4. Howard Hughes Medical Institute, University of California, Berkeley, 94720, California, USA

    Carolyn R Bertozzi

  5. Materials Sciences Division, The Molecular Foundry, Lawrence Berkeley National Laboratory, Berkeley, 94720, California, USA

    Carolyn R Bertozzi

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  1. Marta Fernández-Suárez
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  7. Alice Y Ting
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Contributions

M.F.-S., H.B., L.M.-H. and A.Y.T. designed the research; M.F.-S., H.B., L.M.-H. and K.T.X. performed the research; J.M.B. and C.R.B. provided cyclo-octyne starting material; M.F.-S., H.B. and A.Y.T. analyzed data; M.F.-S. and A.Y.T. wrote the paper.

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Correspondence to Alice Y Ting.

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Supplementary Figures 1–6; Supplementary Methods (PDF 405 kb)

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Fernández-Suárez, M., Baruah, H., Martínez-Hernández, L. et al. Redirecting lipoic acid ligase for cell surface protein labeling with small-molecule probes. Nat Biotechnol 25, 1483–1487 (2007). https://doi.org/10.1038/nbt1355

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