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Expanding the diversity of chemical protein modification allows post-translational mimicry


One of the most important current scientific paradoxes is the economy with which nature uses genes. In all higher animals studied, we have found many fewer genes than we would have previously expected. The functional outputs of the eventual products of genes seem to be far more complex than the more restricted blueprint. In higher organisms, the functions of many proteins are modulated by post-translational modifications (PTMs)1. These alterations of amino-acid side chains lead to higher structural and functional protein diversity and are, therefore, a leading contender for an explanation for this seeming incongruity. Natural protein production methods typically produce PTM mixtures within which function is difficult to dissect or control. Until now it has not been possible to access pure mimics of complex PTMs. Here we report a chemical tagging approach that enables the attachment of multiple modifications to bacterially expressed (bare) protein scaffolds: this approach allows reconstitution of functionally effective mimics of higher organism PTMs. By attaching appropriate modifications at suitable distances in the widely-used LacZ reporter enzyme scaffold, we created protein probes that included sensitive systems for detection of mammalian brain inflammation and disease. Through target synthesis of the desired modification, chemistry provides a structural precision and an ability to retool with a chosen PTM in a manner not available to other approaches. In this way, combining chemical control of PTM with readily available protein scaffolds provides a systematic platform for creating probes of protein–PTM interactions. We therefore anticipate that this ability to build model systems2 will allow some of this gene product complexity to be dissected, with the aim of eventually being able to completely duplicate the patterns of a particular protein’s PTMs from an in vivo assay into an in vitro system.

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Figure 1: Differential multi-site chemical protein modification.
Figure 2: Creating functional mimics of PSGL-1.
Figure 3: Binding of mimics to human P-selectin.
Figure 4: Use of mimics PSGL-LacZ, PSGL*-LacZ and GlcNAc-LacZ as probes.


  1. Wold, F. In vivo chemical modification of proteins (post-translational modification). Annu. Rev. Biochem. 50, 783–814 (1981)

    CAS  Article  Google Scholar 

  2. Davis, B. G. Mimicking posttranslational modifications of proteins. Science 303, 480–482 (2004)

    CAS  Article  Google Scholar 

  3. Dwek, R. A. Glycobiology: Toward understanding the function of sugars. Chem. Rev. 96, 683–720 (1996)

    CAS  Article  Google Scholar 

  4. Davis, B. G. Synthesis of glycoproteins. Chem. Rev. 102, 579–601 (2002)

    CAS  Article  Google Scholar 

  5. Davis, B. G., Lloyd, R. C. & Jones, J. B. Controlled site-selective glycosylation of proteins by a combined site-directed mutagenesis and chemical modification approach. J. Org. Chem. 63, 9614–9615 (1998)

    CAS  Article  Google Scholar 

  6. 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)

    ADS  CAS  Article  Google Scholar 

  7. van Hest, J. C. M., Kiick, K. L. & Tirrell, D. A. Efficient incorporation of unsaturated methionine analogues into proteins in vivo.. J. Am. Chem. Soc. 122, 1282–1288 (2000)

    CAS  Article  Google Scholar 

  8. Fodje, M. N. & Al-Karadaghi, S. Occurrence, conformational features and amino acid propensities for the pi-helix. Protein Eng. 15, 353–358 (2002)

    CAS  Article  Google Scholar 

  9. Cubellis, M. V., Rozzo, C., Montecucchi, P. & Rossi, M. Gene 94, 89–94 (1990)

    CAS  Article  Google Scholar 

  10. Easton, R. L., Patankar, M. S., Clark, G. F., Morris, H. R. & Dell, A. Pregnancy-associated changes in the glycosylation of Tamm-Horsfall glycoprotein. J. Biol. Chem. 275, 21928–21938 (2000)

    CAS  Article  Google Scholar 

  11. Campbell, S. J., Carlotti, F., Hall, P. A., Clark, A. J. & Wolf, C. R. Regulation of the CYP1A1 promoter in transgenic mice: An exquisitely sensitive on-off system for cell specific gene regulation. J. Cell Sci. 109, 2619–2625 (1996)

    CAS  PubMed  Google Scholar 

  12. Rostovtsev, V. V., Green, L. G., Fokin, V. V. & Sharpless, K. B. A stepwise Huisgen cycloaddition process: Copper-catalyzed regioselective ligation of azides and terminal alkynes. Angew. Chem. Int. Edn 41, 2596–2599 (2002)

    CAS  Article  Google Scholar 

  13. Tornøe, C. W., Christensen, C. & Meldal, M. Peptidotriazoles on solid phase: [1,2,3]-triazoles by regiospecific copper(I)-catalyzed 1,3-dipolar cycloadditions of terminal alkynes to azides. J. Org. Chem. 67, 3057–3064 (2002)

    Article  Google Scholar 

  14. Vosseller, K., Sakabe, K., Wells, L. & Hart, G. W. Diverse regulation of protein function by O-GlcNAc: a nuclear and cytoplasmic carbohydrate post-translational modification. Curr. Opin. Chem. Biol. 6, 851–857 (2002)

    CAS  Article  Google Scholar 

  15. Somers, W. S., Tang, J., Shaw, G. D. & Camphausen, R. T. Insights into the molecular basis of leukocyte tethering and rolling revealed by structures of P-and E-selectin bound to SLe(X) and PSGL-1. Cell 103, 467–479 (2000)

    CAS  Article  Google Scholar 

  16. Kansas, G. S. Selectins and their ligands: Current concepts and controversies. Blood 88, 3259 (1996)

    CAS  PubMed  Google Scholar 

  17. Leppänen, A., White, S. P., Helin, J., McEver, R. P. & Cummings, R. D. Binding of glycosulfopeptides to P-selectin requires stereospecific contributions of individual tyrosine sulfate and sugar residues. J. Biol. Chem. 275, 39569–39578 (2000)

    Article  Google Scholar 

  18. Nelson, R. M., Dolich, S., Aruffo, A., Cecconi, O. & Bevilacqua, M. P. Higher-affinity oligosaccharide ligands for E-selectin. J. Clin. Investig. 91, 1157–1166 (1993)

    CAS  Article  Google Scholar 

  19. Barkalow, F. J., Goodman, M. J., Gerritsen, M. E. & Mayadas, T. N. Brain endothelium lack one of two pathways of P-selectin-mediated neutrophil adhesion. Blood 88, 4585–4593 (1996)

    CAS  PubMed  Google Scholar 

  20. Sibson, N. R. et al. MRI detection of early endothelial activation in brain inflammation. Magn. Reson. Med. 51, 248–252 (2004)

    CAS  Article  Google Scholar 

  21. Ferrari, C. C. et al. Reversible demyelination, blood-brain barrier breakdown, and pronounced neutrophil recruitment induced by chronic IL-1 expression in the brain. Am. J. Pathol. 165, 1827–1837 (2004)

    CAS  Article  Google Scholar 

  22. Gotsch, U., Jager, U., Dominis, M. & Vestweber, D. Expression of P-selectin on endothelial cells is upregulated by LPS and TNF-alpha in vivo.. Cell Adhes. Commun. 2, 7–14 (1994)

    CAS  Article  Google Scholar 

  23. Bernardes-Silva, M., Anthony, D. C., Issekutz, A. C. & Perry, V. H. Recruitment of neutrophils across the blood-brain barrier: The role of E- and P-selectins. J. Cereb. Blood Flow Metab. 21, 1115–1124 (2001)

    CAS  Article  Google Scholar 

  24. Schofield, L. & Grau, G. E. Immunological processes in malaria pathogenesis. Nature Rev. Immunol. 5, 722–735 (2005)

    CAS  Article  Google Scholar 

  25. Grau, G. E. et al. Platelet accumulation in brain microvessels in fatal pediatric cerebral malaria. J. Infect. Dis. 187, 461–466 (2003)

    Article  Google Scholar 

  26. Sun, G. et al. Inhibition of platelet adherence to brain microvasculature protects against severe Plasmodium berghei malaria. Infect. Immun. 71, 6553–6561 (2003)

    CAS  Article  Google Scholar 

  27. Khidekel, N., Ficarro, S. B., Peters, E. C. & Hsieh-Wilson, L. C. Exploring the O-GlcNAc proteome: Direct identification of O-GlcNAc-modified proteins from the brain. Proc. Natl Acad. Sci. USA 101, 13132–13137 (2004)

    ADS  CAS  Article  Google Scholar 

  28. Powlesland, A. S. et al. Widely divergent biochemical properties of the complete set of mouse DC-SIGN-related proteins. J. Biol. Chem. 281, 20440–20449 (2006)

    CAS  Article  Google Scholar 

  29. Elliot, S. P., Schmied, R., Gabel, C. A. & Ambron, R. T. An 83-kDa O-GlcNAcglycoprotein is found in the axoplasm and nucleus of Aplysia neurons. J. Neurosci. 13, 2424–2429 (1993)

    CAS  Article  Google Scholar 

  30. Lefebvre, T. et al. Does O-GlcNAc play a role in neurodegenerative diseases? Expert Rev. Proteomics 2, 265–275 (2005)

    CAS  Article  Google Scholar 

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We thank Glycoform (Studentship to S.I.v.K., H.B.K.) and the Danish National Research Science Foundation (Fellowship to H.H.J.) for financial support; K. Drickamer for supply of DC-SIGN-R2 plasmid; and G. Bernardes, I. Davies, P. Wilainam, O. Pearce, K. Doores, A. French, T. P. Hughes, J. Errey, M. Squire, D. Gamblin and E. Scanlan for technical assistance.

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The work described has been patented by the University of Oxford with a view to commercial exploitation. If licensed, this affords royalties in line with standard University practice..

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Supplementary Information

This file contains Supplementary Figures S1-S7 and Legends, Supplementary Methods, Supplementary Tables S1-S3 and Legends, Supplementary Results, Supplementary Discussion, Supplementary Notes and additional references. (PDF 6287 kb)

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van Kasteren, S., Kramer, H., Jensen, H. et al. Expanding the diversity of chemical protein modification allows post-translational mimicry. Nature 446, 1105–1109 (2007).

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