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.

  • Article
  • Published:

Antigen processing by nardilysin and thimet oligopeptidase generates cytotoxic T cell epitopes

Nature Immunology volume 12, pages 45–53 (2011)Cite this article

Subjects

Abstract

Cytotoxic T lymphocytes (CTLs) recognize peptides presented by HLA class I molecules on the cell surface. The C terminus of these CTL epitopes is considered to be produced by the proteasome. Here we demonstrate that the cytosolic endopeptidases nardilysin and thimet oligopeptidase (TOP) complemented proteasome activity. Nardilysin and TOP were required, either together or alone, for the generation of a tumor-specific CTL epitope from PRAME, an immunodominant CTL epitope from Epstein-Barr virus protein EBNA3C, and a clinically important epitope from the melanoma protein MART-1. TOP functioned as C-terminal trimming peptidase in antigen processing, and nardilysin contributed to both the C-terminal and N-terminal generation of CTL epitopes. By broadening the antigenic peptide repertoire, nardilysin and TOP strengthen the immune defense against intracellular pathogens and cancer.

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

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

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

Figure 1: PRA(190–198) is an HLA-A3-presented CTL epitope with a proteasome-independent C terminus.
Figure 2: Nardilysin produces precursors of PRA(190–198) with C-terminal extension.
Figure 3: TOP produces the C terminus of the PRA(190–198) epitope.
Figure 4: Role of nardilysin in HLA class I antigen processing.
Figure 5: The epitope-generating trimming capacity of TOP.
Figure 6: TOP-dependent presentation by HLA-A2 of the MART-1 CTL epitope.

Similar content being viewed by others

Accession codes

Accessions

GenBank/EMBL/DDBJ

References

  1. Rock, K.L., York, I.A. & Goldberg, A.L. Post-proteasomal antigen processing for major histocompatibility complex class I presentation. Nat. Immunol. 5, 670–677 (2004).

    Article  CAS  Google Scholar 

  2. Shastri, N., Cardinaud, S., Schwab, S.R., Serwold, T. & Kunisawa, J. All the peptides that fit: the beginning, the middle, and the end of the MHC class I antigen-processing pathway. Immunol. Rev. 207, 31–41 (2005).

    Article  CAS  Google Scholar 

  3. Yewdell, J.W. Plumbing the sources of endogenous MHC class I peptide ligands. Curr. Opin. Immunol. 19, 79–86 (2007).

    Article  CAS  Google Scholar 

  4. Reits, E. et al. A major role for TPPII in trimming proteasomal degradation products for MHC class I antigen presentation. Immunity 20, 495–506 (2004).

    Article  CAS  Google Scholar 

  5. York, I.A. et al. The cytosolic endopeptidase, thimet oligopeptidase, destroys antigenic peptides and limits the extent of MHC class I antigen presentation. Immunity 18, 429–440 (2003).

    Article  CAS  Google Scholar 

  6. Saric, T., Graef, C.I. & Goldberg, A.L. Pathway for degradation of peptides generated by proteasomes: a key role for thimet oligopeptidase and other metallopeptidases. J. Biol. Chem. 279, 46723–46732 (2004).

    Article  CAS  Google Scholar 

  7. Saveanu, L. et al. Concerted peptide trimming by human ERAP1 and ERAP2 aminopeptidase complexes in the endoplasmic reticulum. Nat. Immunol. 6, 689–697 (2005).

    Article  CAS  Google Scholar 

  8. Craiu, A., Akopian, T., Goldberg, A. & Rock, K.L. Two distinct proteolytic processes in the generation of a major histocompatibility complex class I-presented peptide. Proc. Natl. Acad. Sci. USA 94, 10850–10855 (1997).

    Article  CAS  Google Scholar 

  9. Stoltze, L. et al. Generation of the vesicular stomatitis virus nucleoprotein cytotoxic T lymphocyte epitope requires proteasome-dependent and -independent proteolytic activities. Eur. J. Immunol. 28, 4029–4036 (1998).

    Article  CAS  Google Scholar 

  10. Mo, X.Y., Cascio, P., Lemerise, K., Goldberg, A.L. & Rock, K. Distinct proteolytic processes generate the C and N termini of MHC class I-binding peptides. J. Immunol. 163, 5851–5859 (1999).

    CAS  PubMed  Google Scholar 

  11. Benham, A.M., Gromme, M. & Neefjes, J. Allelic differences in the relationship between proteasome activity and MHC class I peptide loading. J. Immunol. 161, 83–89 (1998).

    CAS  PubMed  Google Scholar 

  12. Luckey, C.J. et al. Proteasomes can either generate or destroy MHC class I epitopes: evidence for nonproteasomal epitope generation in the cytosol. J. Immunol. 161, 112–121 (1998).

    CAS  PubMed  Google Scholar 

  13. Schwarz, K. et al. The selective proteasome inhibitors lactacystin and epoxomicin can be used to either up- or down-regulate antigen presentation at nontoxic doses. J. Immunol. 164, 6147–6157 (2000).

    Article  CAS  Google Scholar 

  14. Luckey, C.J. et al. Differences in the expression of human class I MHC alleles and their associated peptides in the presence of proteasome inhibitors. J. Immunol. 167, 1212–1221 (2001).

    Article  CAS  Google Scholar 

  15. Marcilla, M., Cragnolini, J.J. & Lopez de Castro, J.A. Proteasome-independent HLA-B27 ligands arise mainly from small basic proteins. Mol. Cell. Proteomics 6, 923–938 (2007).

    Article  CAS  Google Scholar 

  16. Seifert, U. et al. An essential role for tripeptidyl peptidase in the generation of an MHC class I epitope. Nat. Immunol. 4, 375–379 (2003).

    Article  CAS  Google Scholar 

  17. Parmentier, N. et al. Production of an antigenic peptide by insulin-degrading enzyme. Nat. Immunol. 11, 449–454 (2010).

    Article  CAS  Google Scholar 

  18. van Endert, P. Role of tripeptidyl peptidase II in MHC class I antigen processing - the end of controversies? Eur. J. Immunol. 38, 609–613 (2008).

    Article  CAS  Google Scholar 

  19. Tenzer, S. et al. Modeling the MHC class I pathway by combining predictions of proteasomal cleavage, TAP transport and MHC class I binding. Cell. Mol. Life Sci. 62, 1025–1037 (2005).

    Article  CAS  Google Scholar 

  20. Kessler, J.H. et al. Efficient identification of novel HLA-A(*)0201-presented cytotoxic T lymphocyte epitopes in the widely expressed tumor antigen PRAME by proteasome-mediated digestion analysis. J. Exp. Med. 193, 73–88 (2001).

    Article  CAS  Google Scholar 

  21. Geier, E. et al. A giant protease with potential to substitute for some functions of the proteasome. Science 283, 978–981 (1999).

    Article  CAS  Google Scholar 

  22. Fumagalli, P. et al. Human NRD convertase: a highly conserved metalloendopeptidase expressed at specific sites during development and in adult tissues. Genomics 47, 238–245 (1998).

    Article  CAS  Google Scholar 

  23. Chow, K.M. et al. Studies on the subsite specificity of rat nardilysin (N-arginine dibasic convertase). J. Biol. Chem. 275, 19545–19551 (2000).

    Article  CAS  Google Scholar 

  24. Chow, K.M. et al. Nardilysin cleaves peptides at monobasic sites. Biochemistry 42, 2239–2244 (2003).

    Article  CAS  Google Scholar 

  25. Knight, C.G., Dando, P.M. & Barrett, A.J. Thimet oligopeptidase specificity: evidence of preferential cleavage near the C-terminus and product inhibition from kinetic analysis of peptide hydrolysis. Biochem. J. 308, 145–150 (1995).

    Article  CAS  Google Scholar 

  26. Oliveira, V. et al. Temperature and salts effects on the peptidase activities of the recombinant metallooligopeptidases neurolysin and thimet oligopeptidase. Eur. J. Biochem. 269, 4326–4334 (2002).

    Article  CAS  Google Scholar 

  27. Oliveira, V. et al. Substrate specificity characterization of recombinant metallo oligo-peptidases thimet oligopeptidase and neurolysin. Biochemistry 40, 4417–4425 (2001).

    Article  CAS  Google Scholar 

  28. van Swieten, P.F. et al. A cell-permeable inhibitor and activity-based probe for the caspase-like activity of the proteasome. Bioorg. Med. Chem. Lett. 17, 3402–3405 (2007).

    Article  CAS  Google Scholar 

  29. Altfeld, M. et al. HLA Alleles associated with delayed progression to AIDS contribute strongly to the initial CD8+ T cell response against HIV-1. PLoS Med. 3, e403 (2006).

    Article  Google Scholar 

  30. Brooks, J.M., Murray, R.J., Thomas, W.A., Kurilla, M.G. & Rickinson, A.B. Different HLA-B27 subtypes present the same immunodominant Epstein-Barr virus peptide. J. Exp. Med. 178, 879–887 (1993).

    Article  CAS  Google Scholar 

  31. Sigman, J.A. et al. Flexibility in substrate recognition by thimet oligopeptidase as revealed by denaturation studies. Biochem. J. 388, 255–261 (2005).

    Article  CAS  Google Scholar 

  32. Kawakami, Y. et al. Identification of the immunodominant peptides of the MART-1 human melanoma antigen recognized by the majority of HLA-A2-restricted tumor infiltrating lymphocytes. J. Exp. Med. 180, 347–352 (1994).

    Article  CAS  Google Scholar 

  33. Romero, P. et al. Cytolytic T lymphocyte recognition of the immunodominant HLA-A*0201-restricted Melan-A/MART-1 antigenic peptide in melanoma. J. Immunol. 159, 2366–2374 (1997).

    CAS  PubMed  Google Scholar 

  34. Le Gall, S., Stamegna, P. & Walker, B.D. Portable flanking sequences modulate CTL epitope processing. J. Clin. Invest. 117, 3563–3575 (2007).

    Article  CAS  Google Scholar 

  35. Schneider, J., Brichard, V., Boon, T., Meyer zum Buschenfelde, K.H. & Wolfel, T. Overlapping peptides of melanocyte differentiation antigen Melan-A/MART- 1 recognized by autologous cytolytic T lymphocytes in association with HLA-B45.1 and HLA-A2.1. Int. J. Cancer 75, 451–458 (1998).

    Article  CAS  Google Scholar 

  36. Silva, C.L., Portaro, F.C., Bonato, V.L., de Camargo, A.C. & Ferro, E.S. Thimet oligopeptidase (EC 3.4.24.15), a novel protein on the route of MHC class I antigen presentation. Biochem. Biophys. Res. Commun. 255, 591–595 (1999).

    Article  CAS  Google Scholar 

  37. Portaro, F.C. et al. Thimet oligopeptidase and the stability of MHC class I epitopes in macrophage cytosol. Biochem. Biophys. Res. Commun. 255, 596–601 (1999).

    Article  CAS  Google Scholar 

  38. Saric, T. et al. Major histocompatibility complex class I-presented antigenic peptides are degraded in cytosolic extracts primarily by thimet oligopeptidase. J. Biol. Chem. 276, 36474–36481 (2001).

    Article  CAS  Google Scholar 

  39. Kim, S.I., Pabon, A., Swanson, T.A. & Glucksman, M.J. Regulation of cell-surface major histocompatibility complex class I expression by the endopeptidase EC3.4.24.15 (thimet oligopeptidase). Biochem. J. 375, 111–120 (2003).

    Article  CAS  Google Scholar 

  40. Berti, D.A. et al. Analysis of intracellular substrates and products of thimet oligopeptidase (EC 3.4.24.15) in human embryonic kidney 293 cells. J. Biol. Chem. 284, 14105–14116 (2009).

    Article  CAS  Google Scholar 

  41. Herberts, C.A. et al. Cutting edge: HLA-B27 acquires Many N-terminal dibasic peptides: coupling cytosolic peptide stability to antigen presentation. J. Immunol. 176, 2697–2701 (2006).

    Article  CAS  Google Scholar 

  42. Csuhai, E., Chen, G. & Hersh, L.B. Regulation of N-arginine dibasic convertase activity by amines: putative role of a novel acidic domain as an amine binding site. Biochemistry 37, 3787–3794 (1998).

    Article  CAS  Google Scholar 

  43. Mulder, A. et al. Human monoclonal HLA antibodies reveal interspecies crossreactive swine MHC class I epitopes relevant for xenotransplantation. Mol. Immunol. 47, 809–815 (2010).

    Article  CAS  Google Scholar 

  44. Kessler, J.H. et al. Competition-based cellular peptide binding assays for 13 prevalent HLA class I alleles using fluorescein-labeled synthetic peptides. Hum. Immunol. 64, 245–255 (2003).

    Article  CAS  Google Scholar 

  45. Kessler, B.M. et al. Extended peptide-based inhibitors efficiently target the proteasome and reveal overlapping specificities of the catalytic beta-subunits. Chem. Biol. 8, 913–929 (2001).

    Article  CAS  Google Scholar 

  46. Balow, R.M., Tomkinson, B., Ragnarsson, U. & Zetterqvist, O. Purification, substrate specificity, and classification of tripeptidyl peptidase II. J. Biol. Chem. 261, 2409–2417 (1986).

    CAS  PubMed  Google Scholar 

  47. Tomkinson, B. & Zetterqvist, O. Immunological cross-reactivity between human tripeptidyl peptidase II and fibronectin. Biochem. J. 267, 149–154 (1990).

    Article  CAS  Google Scholar 

  48. Roelse, J., Gromme, M., Momburg, F., Hammerling, G. & Neefjes, J. Trimming of TAP-translocated peptides in the endoplasmic reticulum and in the cytosol during recycling. J. Exp. Med. 180, 1591–1597 (1994).

    Article  CAS  Google Scholar 

  49. Neisig, A. et al. Major differences in transporter associated with antigen presentation (TAP)-dependent translocation of MHC class I- presentable peptides and the effect of flanking sequences. J. Immunol. 154, 1273–1279 (1995).

    CAS  PubMed  Google Scholar 

  50. Androlewicz, M.J. & Cresswell, P. Human transporters associated with antigen processing possess a promiscuous peptide-binding site. Immunity 1, 7–14 (1994).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank T. Dannenberg and K. Textoris-Taube for technical assistance. Supported by the Dutch Cancer Society (UL 2005-3245) and Stichting Vanderes.

Author information

Author notes

  1. Peter A van Veelen, Ferry Ossendorp and Cornelis J M Melief: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Immunohematology and Blood Transfusion, Leiden University Medical Center, Leiden, The Netherlands

    Jan H Kessler, Selina Khan, Sandra A Bres-Vloemans, Arnoud de Ru, Nadine van Montfoort, Kees L M C Franken, Willemien E Benckhuijsen, Arend Mulder, Ilias I N Doxiadis, Jan W Drijfhout, Peter A van Veelen, Ferry Ossendorp & Cornelis J M Melief

  2. Institut für Biochemie-Charité, Medical Faculty of the Humboldt University Berlin, Berlin, Germany.,

    Ulrike Seifert & Peter M Kloetzel

  3. Ragon Institute of MGH, MIT and Harvard, Boston, Massachusetts, USA.,

    Sylvie Le Gall

  4. Department of Molecular and Cellular Biochemistry, and Center for Structural Biology, University of Kentucky, Lexington, Kentucky, USA.,

    K Martin Chow, Kallol Ray, David W Rodgers & Louis B Hersh

  5. Department of Dermatology, University Clinics Essen, Essen, Germany.,

    Annette Paschen

  6. Cancer Research UK Institute for Cancer Studies, University of Birmingham, Birmingham, UK

    Jill M Brooks

  7. Department of Clinical Oncology, Leiden University Medical Center, Leiden, The Netherlands.,

    Thorbald van Hall

  8. Leiden Institute of Chemistry, Leiden University, Leiden, The Netherlands.,

    Paul F van Swieten & Hermen S Overkleeft

  9. Laboratory of Biochemical Neuroendocrinology, Clinical Research Institute of Montreal, Montreal, Canada

    Annik Prat

  10. Department of Medical Biochemistry and Microbiology, Uppsala University, Uppsala, Sweden.,

    Birgitta Tomkinson

  11. Division of Cell Biology, The Netherlands Cancer Institute, Amsterdam, The Netherlands

    Jacques Neefjes

Authors
  1. Jan H Kessler
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Selina Khan
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Ulrike Seifert
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Sylvie Le Gall
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. K Martin Chow
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Annette Paschen
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Sandra A Bres-Vloemans
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Arnoud de Ru
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Nadine van Montfoort
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Kees L M C Franken
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Willemien E Benckhuijsen
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Jill M Brooks
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. Thorbald van Hall
    View author publications

    You can also search for this author in PubMed Google Scholar

  14. Kallol Ray
    View author publications

    You can also search for this author in PubMed Google Scholar

  15. Arend Mulder
    View author publications

    You can also search for this author in PubMed Google Scholar

  16. Ilias I N Doxiadis
    View author publications

    You can also search for this author in PubMed Google Scholar

  17. Paul F van Swieten
    View author publications

    You can also search for this author in PubMed Google Scholar

  18. Hermen S Overkleeft
    View author publications

    You can also search for this author in PubMed Google Scholar

  19. Annik Prat
    View author publications

    You can also search for this author in PubMed Google Scholar

  20. Birgitta Tomkinson
    View author publications

    You can also search for this author in PubMed Google Scholar

  21. Jacques Neefjes
    View author publications

    You can also search for this author in PubMed Google Scholar

  22. Peter M Kloetzel
    View author publications

    You can also search for this author in PubMed Google Scholar

  23. David W Rodgers
    View author publications

    You can also search for this author in PubMed Google Scholar

  24. Louis B Hersh
    View author publications

    You can also search for this author in PubMed Google Scholar

  25. Jan W Drijfhout
    View author publications

    You can also search for this author in PubMed Google Scholar

  26. Peter A van Veelen
    View author publications

    You can also search for this author in PubMed Google Scholar

  27. Ferry Ossendorp
    View author publications

    You can also search for this author in PubMed Google Scholar

  28. Cornelis J M Melief
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

J.H.K. conceived of the study, coordinated the work, designed, did and analyzed most experiments and wrote the manuscript with major input from C.J.M.M. and minor input from other authors; C.J.M.M., F.O. and P.A.v.V. provided intellectual input; S.K. did the experiments and analyses in Figure 2b and immunoblot analysis; K.M.C., L.B.H. and A. Prat contributed to the experiments about nardilysin; D.W.R., K.R., U.S. and A. Paschen contributed to the experiments about TOP; P.M.K., U.S. and B.T. contributed to the experiments about TPPII; H.S.O. and P.F.v.S. contributed to the experiments about the proteasome; J.N. and T.v.H. contributed to the experiments about TAP; N.v.M., U.S., A. Paschen, S.L.G. and J.M.B. contributed to CTL experiments; J.W.D., F.O., J.N., S.K. and W.E.B. contributed to substrate design and synthesis; S.A.B.-V. and K.L.M.C.F. contributed to the molecular biology; A.M. and I.I.N.D. made HLA class I mAbs and cell lines expressing a single HLA class I allele; and P.A.v.V. and A.d.R. did mass spectrometry.

Corresponding author

Correspondence to Jan H Kessler.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–12 and Supplementary Tables 1–2 (PDF 3479 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Kessler, J., Khan, S., Seifert, U. et al. Antigen processing by nardilysin and thimet oligopeptidase generates cytotoxic T cell epitopes. Nat Immunol 12, 45–53 (2011). https://doi.org/10.1038/ni.1974

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/ni.1974

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