Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Fluorogenic probes for monitoring peptide binding to class II MHC proteins in living cells

Nature Chemical Biology volume 3, pages 222–228 (2007)Cite this article

Abstract

A crucial step in the immune response is the binding of antigenic peptides to major histocompatibility complex (MHC) proteins. Class II MHC proteins present their bound peptides to CD4+ T cells, thereby helping to activate both the humoral and the cellular arms of the adaptive immune response. Peptide loading onto class II MHC proteins is regulated temporally, spatially and developmentally in antigen-presenting cells1. To help visualize these processes, we have developed a series of novel fluorogenic probes that incorporate the environment-sensitive amino acid analogs 6-N,N-dimethylamino-2-3-naphthalimidoalanine and 4-N,N-dimethylaminophthalimidoalanine. Upon binding to class II MHC proteins these fluorophores show large changes in emission spectra, quantum yield and fluorescence lifetime. Peptides incorporating these fluorophores bind specifically to class II MHC proteins on antigen-presenting cells and can be used to follow peptide binding in vivo. Using these probes we have tracked a developmentally regulated cell-surface peptide-binding activity in primary human monocyte-derived dendritic cells.

This is a preview of subscription content, access via your institution

Relevant articles

Open Access articles citing this article.

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Learn more

Prices may be subject to local taxes which are calculated during checkout

Figure 1: 4-DAPA and 6-DMNA can be modeled into the P1 pocket of DR1 without major distortion.
Figure 2: Spectral properties of 6-DMNA and 4-DAPA peptides free in solution and bound to DR1.
Figure 3: Structural and functional characterization of (4-DAPA)-HA peptide.
Figure 4: (6-DMNA)-peptide binds specifically to DR molecules expressed on live cells.
Figure 5: (6-DMNA)-peptide binds to human monocyte-derived DCs in a maturation-dependent manner.

Accession codes

Accessions

Protein Data Bank

References

  1. Trombetta, E.S. & Mellman, I. Cell biology of antigen processing in vitro and in vivo. Annu. Rev. Immunol. 23, 975–1028 (2005).

    Article  CAS  Google Scholar 

  2. McFarland, B.J. & Beeson, C. Binding interactions between peptides and proteins of the class II major histocompatibility complex. Med. Res. Rev. 22, 168–203 (2002).

    Article  CAS  Google Scholar 

  3. Jardetzky, T.S. et al. Peptide binding to HLA-DR1: a peptide with most residues substituted to alanine retains MHC binding. EMBO J. 9, 1797–1803 (1990).

    Article  CAS  Google Scholar 

  4. Sato, A.K. et al. Determinants of the peptide-induced conformational change in the human class II major histocompatibility complex protein HLA-DR1. J. Biol. Chem. 275, 2165–2173 (2000).

    Article  CAS  Google Scholar 

  5. Stern, L.J. et al. Crystal structure of the human class II MHC protein HLA-DR1 complexed with an influenza virus peptide. Nature 368, 215–221 (1994).

    Article  CAS  Google Scholar 

  6. Cohen, B.E. et al. Probing protein electrostatics with a synthetic fluorescent amino acid. Science 296, 1700–1703 (2002).

    Article  CAS  Google Scholar 

  7. Nitz, M., Mezo, A.R., Ali, M.H. & Imperiali, B. Enantioselective synthesis and application of the highly fluorescent and environment-sensitive amino acid 6-(2-dimethylaminonaphthoyl) alanine (DANA). Chem. Commun. (Camb.) 1912–1913 (2002).

  8. Vazquez, M.E., Rothman, D.M. & Imperiali, B. A new environment-sensitive fluorescent amino acid for Fmoc-based solid phase peptide synthesis. Org. Biomol. Chem. 2, 1965–1966 (2004).

    Article  CAS  Google Scholar 

  9. Vazquez, M.E., Blanco, J.B. & Imperiali, B. Photophysics and biological applications of the environment-sensitive fluorophore 6-N,N-dimethylamino-2,3-naphthalimide. J. Am. Chem. Soc. 127, 1300–1306 (2005).

    Article  CAS  Google Scholar 

  10. Chen, H. et al. [Aladan3]TIPP: a fluorescent δ-opioid antagonist with high δ-receptor binding affinity and δ selectivity. Biopolymers 80, 325–331 (2005).

    Article  CAS  Google Scholar 

  11. Saroja, G., Soujanya, T., Ramachandram, B. & Samanta, A. 4-Aminophthalimide derivatives as environment-sensitive probes. J. Fluoresc. 8, 405–410 (1998).

    Article  CAS  Google Scholar 

  12. Hammer, J. et al. High-affinity binding of short peptides to major histocompatibility complex class II molecules by anchor combinations. Proc. Natl. Acad. Sci. USA 91, 4456–4460 (1994).

    Article  CAS  Google Scholar 

  13. Roche, P.A. & Cresswell, P. High-affinity binding of an influenza hemagglutinin-derived peptide to purified HLA-DR. J. Immunol. 144, 1849–1856 (1990).

    CAS  PubMed  Google Scholar 

  14. Denzin, L.K., Robbins, N.F., Carboy-Newcomb, C. & Cresswell, P. Assembly and intracellular transport of HLA-DM and correction of the class II antigen-processing defect in T2 cells. Immunity 1, 595–606 (1994).

    Article  CAS  Google Scholar 

  15. Potolicchio, I. et al. Conformational variation of surface class II MHC proteins during myeloid dendritic cell differentiation accompanies structural changes in lysosomal MIIC. J. Immunol. 175, 4935–4947 (2005).

    Article  CAS  Google Scholar 

  16. Pierre, P. & Mellman, I. Developmental regulation of invariant chain proteolysis controls MHC class II trafficking in mouse dendritic cells. Cell 93, 1135–1145 (1998).

    Article  CAS  Google Scholar 

  17. Turley, S.J. et al. Transport of peptide-MHC class II complexes in developing dendritic cells. Science 288, 522–527 (2000).

    Article  CAS  Google Scholar 

  18. Inaba, K. et al. The formation of immunogenic major histocompatibility complex class II-peptide ligands in lysosomal compartments of dendritic cells is regulated by inflammatory stimuli. J. Exp. Med. 191, 927–936 (2000).

    Article  CAS  Google Scholar 

  19. Cella, M., Engering, A., Pinet, V., Pieters, J. & Lanzavecchia, A. Inflammatory stimuli induce accumulation of MHC class II complexes on dendritic cells. Nature 388, 782–787 (1997).

    Article  CAS  Google Scholar 

  20. Colledge, L., Bennett, C.L., Reay, P.A. & Blackburn, C.C. Rapid constitutive generation of a specific peptide-MHC class II complex from intact exogenous protein in immature murine dendritic cells. Eur. J. Immunol. 32, 3246–3255 (2002).

    Article  CAS  Google Scholar 

  21. Veeraswamy, R.K., Cella, M., Colonna, M. & Unanue, E.R. Dendritic cells process and present antigens across a range of maturation states. J. Immunol. 170, 5367–5372 (2003).

    Article  CAS  Google Scholar 

  22. Wilson, N.S., El-Sukkari, D. & Villadangos, J.A. Dendritic cells constitutively present self antigens in their immature state in vivo and regulate antigen presentation by controlling the rates of MHC class II synthesis and endocytosis. Blood 103, 2187–2195 (2004).

    Article  CAS  Google Scholar 

  23. Southwood, S. et al. Several common HLA-DR types share largely overlapping peptide binding repertoires. J. Immunol. 160, 3363–3373 (1998).

    CAS  PubMed  Google Scholar 

  24. Vazquez, M.E., Nitz, M., Stehn, J., Yaffe, M.B. & Imperiali, B. Fluorescent caged phosphoserine peptides as probes to investigate phosphorylation-dependent protein associations. J. Am. Chem. Soc. 125, 10150–10151 (2003).

    Article  CAS  Google Scholar 

  25. Vazquez, M.E. et al. 6-N,N-dimethylamino-2,3-naphthalimide: a new environment-sensitive fluorescent probe in δ- and μ-selective opioid peptides. J. Med. Chem. 49, 3653–3658 (2006).

    Article  CAS  Google Scholar 

  26. Stern, L.J. & Wiley, D.C. The human class II MHC protein HLA-DR1 assembles as empty α β heterodimers in the absence of antigenic peptide. Cell 68, 465–477 (1992).

    Article  CAS  Google Scholar 

  27. Riberdy, J.M. & Cresswell, P. The antigen-processing mutant T2 suggests a role for MHC-linked genes in class II antigen presentation. J. Immunol. 148, 2586–2590 (1992).

    CAS  PubMed  Google Scholar 

  28. Alberti, S., Parks, D.R. & Herzenberg, L.A. A single laser method for subtraction of cell autofluorescence in flow cytometry. Cytometry 8, 114–119 (1987).

    Article  CAS  Google Scholar 

  29. Parks, D.R., Roederer, M. & Moore, W.A. A new “Logicle” display method avoids deceptive effects of logarithmic scaling for low signals and compensated data. Cytometry A 69, 541–551 (2006).

    Article  Google Scholar 

  30. DeLano, W.L. The PyMOL Molecular Graphics System (DeLano Scientific, San Carlos, California, USA, 2002).

    Google Scholar 

Download references

Acknowledgements

We would like to thank M. Calvo-Calle for performing T-cell activation assays, R. Konz for assistance with flow cytometry experimentation and data interpretation, E. Lukovic and C. Painter for peptide synthesis, I. Strug for characterization of eluted peptides, E. Schreiter and Z. Zavala-Ruiz for helpful discussions, L. Lu for preparation of HLA-DR, J. Bill for the HA1.7 hybridoma, and the staff at the National Synchrotron Light Source (NSLS). Diffraction data for this study were measured at beamline X25 of the NSLS, support for which comes principally from the Office of Biological and Environmental Research and the Office of Basic Energy Sciences of the US Department of Energy, and from the National Center for Research Resources of the US National Institutes of Health. This work was supported by NIH-AI38996 (L.J.S.) and NSF CHE-0414243 (B.I.).

Author information

Author notes

  1. Prasanna Venkatraman

    Present address: Present address: the Advanced Centre for Treatment, Research and Education in Cancer, Kharghar, Navi Mumbai 410208, India.,

Authors and Affiliations

  1. Department of Pathology, University of Massachusetts Medical School, 55 Lake Ave. North, Worcester, 01655, Massachusetts, USA

    Prasanna Venkatraman, Sriram Chitta & Lawrence J Stern

  2. Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, 55 Lake Ave. North, Worcester, 01655, Massachusetts, USA

    Tina T Nguyen, Osman Bilsel & Lawrence J Stern

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

    Matthieu Sainlos & Barbara Imperiali

  4. Department of Biology, 77 Massachusetts Ave., Massachusetts Institute of Technology, Cambridge, 02139, Massachusetts, USA

    Barbara Imperiali

Authors
  1. Prasanna Venkatraman
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Tina T Nguyen
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Matthieu Sainlos
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Osman Bilsel
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Sriram Chitta
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Barbara Imperiali
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Lawrence J Stern
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

P.V. performed the biochemical, cellular and spectroscopic studies, M.S. synthesized the fluorogenic amino acids and performed the chemical stability studies, T.N. determined the crystal structure, O.B. and S.C. performed biophysical and immunological assays, and P.V., M.S., B.I. and L.J.S wrote the manuscript.

Corresponding authors

Correspondence to Barbara Imperiali or Lawrence J Stern.

Ethics declarations

Competing interests

There is a patent application pending (filed 13 April 2005) for the environment-sensitive amino acids originally introduced in J. Am. Chem. Soc. 127, 1300–1306 (2005).

Supplementary information

Supplementary Fig. 1

Absorbance and fluorescence spectra. (PDF 149 kb)

Supplementary Fig. 2

Peptide binding takes place primarily on the cell surface. (PDF 83 kb)

Supplementary Fig. 3

Open forms of (4-DAPA) and (4-DMNA) peptides. (PDF 267 kb)

Supplementary Table 1

Binding affinity of fluorogenic peptides. (PDF 27 kb)

Supplementary Table 2

(6-DMNA)-RSMA4L fluorescence intensity in different solvents. (PDF 44 kb)

Supplementary Table 3

Fluorescence lifetimes for free and DR-bound peptides. (PDF 74 kb)

Supplementary Table 4

Data collection and refinement statistics (molecular replacement). (PDF 71 kb)

Supplementary Methods

Preparation and analysis of ring-open forms; crystal structure; MHC-peptide binding assay; T-cell activation; fluorescence lifetime measurement. (PDF 60 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Venkatraman, P., Nguyen, T., Sainlos, M. et al. Fluorogenic probes for monitoring peptide binding to class II MHC proteins in living cells. Nat Chem Biol 3, 222–228 (2007). https://doi.org/10.1038/nchembio868

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nchembio868

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing