Skip to main content

Advertisement

SpringerLink
Log in

Probing the impact of GFP tagging on Robo1-heparin interaction

  • Published:
Glycoconjugate Journal Aims and scope Submit manuscript

Abstract

Green fluorescent proteins (GFPs) and their derivatives are widely used as markers to visualize cells, protein localizations in in vitro and in vivo studies. The use of GFP fusion protein for visualization is generally thought to have negligible effects on cellular function. However, a number of reports suggest that the use of GFP may impact the biological activity of these proteins. Heparin is a glycosaminoglycan (GAG) that interacts with a number of proteins mediating diverse patho-physiological processes. In the heparin-based interactome studies, heparin-binding proteins are often prepared as GFP fusion proteins. In this report, we use surface plasmon resonance (SPR) spectroscopy to study the impact of the GFP tagging on the binding interaction between heparin and a heparin-binding protein, the Roundabout homolog 1 (Robo1). SPR reveals that heparin binds with higher affinity to Robo1 than GFP-tagged Robo1 and through a different kinetic mechanism. A conformational change is observed in the heparin-Robo1 interaction, but not in the heparin-Robo1-GFP interaction. Furthermore the GFP-tagged Robo1 requires a shorter (hexasaccharide) than the tag-free Robo1 (octadecasaccharide). These data demonstrate that GFP tagging can reduce the binding affinity of Robo1 to heparin and hinder heparin binding-induced Robo1 conformation change.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

Abbreviations

GFPs:

Green fluorescent proteins

SPR:

Surface plasmon resonance

GAG:

Glycosaminoglycan

Robo1:

Roundabout homolog 1

HEPES:

4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid

HS:

Heparan sulfate

RU:

Resonance unit

dp:

Degree of polymerization

References

  1. Shimomura, O., Johnson, F.H., Saiga, Y.: Extraction, purification and properties of aequorin, a bioluminescent protein from the luminous hydromedusan, Aequorea. J. Cell. Comp. Physiol. 59, 223–239 (1962)

    Article  CAS  PubMed  Google Scholar 

  2. Kremers, G.J., Gilbert, S.G., Cranfill, P.J., Davidson, M.W., Piston, D.W.: Fluorescent proteins at a glance. J. Cell Sci. 124, 157–160 (2011)

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  3. Ormo, M., Cubitt, A.B.., Kallio, K., Gross, L.A., Tsien, R.Y., Remington, S.J.: Crystal structure of the Aequorea victoria green fluorescent protein. Science 273, 1392–1395 (1996)

    Article  CAS  PubMed  Google Scholar 

  4. Remington, S.J.: Fluorescent proteins: maturation, photochemistry and photophysics. Curr. Opin. Struct. Biol. 16, 714–721 (2006)

    Article  CAS  PubMed  Google Scholar 

  5. Yarbrough, D., Wachter, R.M., Kallio, K., Matz, M.V., Remington, S.J.: Refined crystal structure of DsRed, a red fluorescent protein from coral, at 2.0-A resolution. Proc. Natl. Acad. Sci. U. S. A. 98, 462–467 (2001)

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  6. Petersen, J., Wilmann, P.G., Beddoe, T., Oakley, A.J., Devenish, R.J., Prescott, M., Rossjohn, J.: The 2.0-A crystal structure of eqFP611, a far red fluorescent protein from the sea anemone Entacmaea quadricolor. J. Biol. Chem. 278, 44626–44631 (2003)

    Article  CAS  PubMed  Google Scholar 

  7. Chalfie, M., Tu, Y., Euskirchen, G., Ward, W.W., Prasher, D.C.: Green fluorescent protein as a marker for gene expression. Science 263, 802–805 (1994)

    Article  CAS  PubMed  Google Scholar 

  8. Davidson, M.W., Campbell, R.E.: Engineered fluorescent proteins: innovations and applications. Nat. Methods 6, 713–717 (2009)

    Article  CAS  PubMed  Google Scholar 

  9. Agbulut, O., Coirault, C., Niederlander, N., Huet, A., Vicart, P., Hagege, A., Puceat, M., Menasche, P.: GFP expression in muscle cells impairs actin-myosin interactions: implications for cell therapy. Nat. Methods 3, 331 (2006)

    Article  CAS  PubMed  Google Scholar 

  10. Agbulut, O., Huet, A., Niederlander, N., Puceat, M., Menasche, P., Coirault, C.: Green fluorescent protein impairs actin-myosin interactions by binding to the actin-binding site of myosin. J. Biol. Chem. 282, 10465–10471 (2007)

    Article  CAS  PubMed  Google Scholar 

  11. Baens, M., Noels, H., Broeckx, V., Hagens, S., Fevery, S., Billiau, A.D., Vankelecom, H., Marynen, P.: The dark side of EGFP: defective polyubiquitination. PLoS One 1, e54 (2006)

    Article  PubMed Central  PubMed  Google Scholar 

  12. Liu, H.S., Jan, M.S., Chou, C.K., Chen, P.H., Ke, N.J.: Is green fluorescent protein toxic to the living cells? Biochem. Biophys. Res. Commun. 260, 712–717 (1999)

    Article  CAS  PubMed  Google Scholar 

  13. Mak, G.W., Wong, C.H., Tsui, S.K.: Green fluorescent protein induces the secretion of inflammatory cytokine interleukin-6 in muscle cells. Anal. Biochem. 362, 296–298 (2007)

    Article  CAS  PubMed  Google Scholar 

  14. Koelsch, K.A., Wang, Y., Maier-Moore, J.S., Sawalha, A.H., Wren, J.D.: GFP affects human T cell activation and cytokine production following in vitro stimulation. PLoS One 8, e50068 (2013)

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  15. Huang, W.Y., Aramburu, J., Douglas, P.S., Izumo, S.: Transgenic expression of green fluorescence protein can cause dilated cardiomyopathy. Nat. Med. 6, 482–483 (2000)

    Article  CAS  PubMed  Google Scholar 

  16. Krestel, H.E., Mihaljevic, A.L., Hoffman, D.A., Schneider, A.: Neuronal co-expression of EGFP and beta-galactosidase in mice causes neuropathology and premature death. Neurobiol. Dis. 17, 310–318 (2004)

    Article  CAS  PubMed  Google Scholar 

  17. Capila, I., Linhardt, R.J.: Heparin-protein interactions. Angew. Chem. Int. Ed. Engl. 41, 391–412 (2002)

    Article  PubMed  Google Scholar 

  18. Hacker, U., Nybakken, K., Perrimon, N.: Heparan sulphate proteoglycans: the sweet side of development. Nat. Rev. Mol. Cell Biol. 6, 530–541 (2005)

    Article  PubMed  Google Scholar 

  19. Parish, C.R.: The role of heparan sulphate in inflammation. Nat. Rev. Immunol. 6, 633–643 (2006)

    Article  CAS  PubMed  Google Scholar 

  20. Powell, A.K., Yates, E.A., Fernig, D.G., Turnbull, J.E.: Interactions of heparin/heparan sulfate with proteins: appraisal of structural factors and experimental approaches. Glycobiology 14, 17R–30R (2004)

    Article  CAS  PubMed  Google Scholar 

  21. Sasisekharan, R., Raman, R., Prabhakar, V.: Glycomics approach to structure-function relationships of glycosaminoglycans. Annu. Rev. Biomed. Eng. 8, 181–231 (2006)

    Article  CAS  PubMed  Google Scholar 

  22. Qiu, H., Jiang, J.L., Liu, M., Huang, X., Ding, S.J., Wang, L.: Quantitative phosphoproteomics analysis reveals broad regulatory role of heparan sulfate on endothelial signaling. Mol. Cell. Proteomics 12, 2160–2173 (2013)

    Article  CAS  PubMed  Google Scholar 

  23. Wang, L., Fuster, M., Sriramarao, P., Esko, J.D.: Endothelial heparan sulfate deficiency impairs L-selectin- and chemokine-mediated neutrophil trafficking during inflammatory responses. Nat. Immunol. 6, 902–910 (2005)

    Article  CAS  PubMed  Google Scholar 

  24. Brose, K., Bland, K.S., Wang, K.H., Arnott, D., Henzel, W., Goodman, C.S., Tessier-Lavigne, M., Kidd, T.: Slit proteins bind Robo receptors and have an evolutionarily conserved role in repulsive axon guidance. Cell 96, 795–806 (1999)

    Article  CAS  PubMed  Google Scholar 

  25. Jones, C.A., et al.: Robo4 stabilizes the vascular network by inhibiting pathologic angiogenesis and endothelial hyperpermeability. Nat. Med. 14, 448–453 (2008)

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  26. Zhang, B., Dietrich, U.M., Geng, J.G., Bicknell, R., Esko, J.D., Wang, L.: Repulsive axon guidance molecule Slit3 is a novel angiogenic factor. Blood 114, 4300–4309 (2009)

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  27. Condac, E., et al.: The C-terminal fragment of axon guidance molecule Slit3 binds heparin and neutralizes heparin's anticoagulant activity. Glycobiology 22, 1183–1192 (2012)

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  28. Barb, A.W., Meng, L., Gao, Z., Johnson, R.W., Moremen, K.W., Prestegard, J.H.: NMR characterization of immunoglobulin G Fc glycan motion on enzymatic sialylation. Biochemistry 51, 4618–4626 (2012)

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  29. Vandersall-Nairn, A.S., Merkle, R.K., O'Brien, K., Oeltmann, T.N., Moremen, K.W.: Cloning, expression, purification, and characterization of the acid alpha-mannosidase from Trypanosoma cruzi. Glycobiology 8, 1183–1194 (1998)

    Article  CAS  PubMed  Google Scholar 

  30. Beckett, D., Kovaleva, E., Schatz, P.J.: A minimal peptide substrate in biotin holoenzyme synthetase-catalyzed biotinylation. Protein Sci. 8, 921–929 (1999)

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  31. Pedelacq, J.D., Cabantous, S., Tran, T., Terwilliger, T.C., Waldo, G.S.: Engineering and characterization of a superfolder green fluorescent protein. Nat. Biotechnol. 24, 79–88 (2006)

    Article  CAS  PubMed  Google Scholar 

  32. Hernaiz, M., Liu, J., Rosenberg, R.D., Linhardt, R.J.: Enzymatic modification of heparan sulfate on a biochip promotes its interaction with antithrombin III. Biochem. Biophys. Res. Commun. 276, 292–297 (2000)

    Article  CAS  PubMed  Google Scholar 

  33. Prince, R.N., Schreiter, E.R., Zou, P., Wiley, H.S., Ting, A.Y., Lee, R.T., Lauffenburger, D.A.: The heparin-binding domain of HB-EGF mediates localization to sites of cell-cell contact and prevents HB-EGF proteolytic release. J. Cell Sci. 123, 2308–2318 (2010)

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  34. Schmidt, M., Govindasamy, L., Afione, S., Kaludov, N., Agbandje-McKenna, M., Chiorini, J.A.: Molecular characterization of the heparin-dependent transduction domain on the capsid of a novel adeno-associated virus isolate, AAV(VR-942). J. Virol. 82, 8911–8916 (2008)

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  35. Tavaré, J.M., Fletcher, L.M., Welsh, G.I.: Using green fluorescent protein to study intracellular signalling. J. Endocrinol. 170, 297–306 (2001)

    Article  PubMed  Google Scholar 

  36. Fukuhara, N., Howitt, J.A., Hussain, S.A., Hohenester, E.: Structural and functional analysis of slit and heparin binding to immunoglobulin-like domains 1 and 2 of Drosophila Robo. J. Biol. Chem. 283, 16226–16234 (2008)

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  37. Olson, S.T., Bjork, I., Sheffer, R., Craig, P.A., Shore, J.D., Choay, J.: Role of the antithrombin-binding pentasaccharide in heparin acceleration of antithrombin-proteinase reactions. Resolution of the antithrombin conformational change contribution to heparin rate enhancement. J. Biol. Chem. 267, 12528–12538 (1992)

    CAS  PubMed  Google Scholar 

  38. Zhang, F., Moniz, H.A., Walcott, B., Moremen, K.W., Linhardt, R.J., Wang, L.: Characterization of the interaction between Robo1 and heparin/glycosaminoglycans. Biochemie 95, 2345–2353 (2013)

    Article  CAS  Google Scholar 

  39. Futamura, M., Dhanasekaran, P., Handa, T., Phillips, M.C., Lund-Katz, S., Saito, H.: Two-step mechanism of binding of apolipoprotein E to heparin: implications for the kinetics of apolipoprotein E-heparan sulfate proteoglycan complex formation on cell surfaces. J. Biol. Chem. 280, 5414–5422 (2005)

    Article  CAS  PubMed  Google Scholar 

  40. Schlessinger, A., Rost, B.: Protein flexibility and rigidity predicted from sequence. Proteins 61, 115–126 (2005)

    Article  CAS  PubMed  Google Scholar 

  41. Schlessinger, A., Punta, M., Rost, B.: Natively unstructured regions in proteins identified from contact predictions. Bioinformatics 23, 2376–2384 (2007)

    Article  CAS  PubMed  Google Scholar 

  42. Schlessinger, A., Punta, M., Yachdav, G., Kajan, L., Rost, B.: Improved disorder prediction by combination of orthogonal approaches. PLoS ONE 4, e4433 (2009)

    Article  PubMed Central  PubMed  Google Scholar 

Download references

Acknowledgments

This work was supported by grants from the National Institutes of Health in the form of GM-38060 to R.J.L. and NIH R01HL093339 (L.W.), RR005351/GM103390 (L.W. and K.M.)

Author information

Authors and Affiliations

  1. Department of Chemical and Biological Engineering, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY, 12180, USA

    Fuming Zhang & Robert J. Linhardt

  2. Department of Biochemistry and Molecular Biology, Complex Carbohydrate Research Center, University of Georgia, Athens, GA, 30602, USA

    Heather A. Moniz, Kelley W. Moremen & Lianchun Wang

  3. Department of Chemistry and Chemical Biology, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY, 12180, USA

    Benjamin Walcott & Robert J. Linhardt

  4. Departments of Biology and Biomedical Engineering, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY, 12180, USA

    Robert J. Linhardt

Authors
  1. Fuming Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Heather A. Moniz
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Benjamin Walcott
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Kelley W. Moremen
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Lianchun Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Robert J. Linhardt
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Fuming Zhang or Robert J. Linhardt.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Zhang, F., Moniz, H.A., Walcott, B. et al. Probing the impact of GFP tagging on Robo1-heparin interaction. Glycoconj J 31, 299–307 (2014). https://doi.org/10.1007/s10719-014-9522-1

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10719-014-9522-1

Keywords

Search

Navigation