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An IFN-γ–induced aminopeptidase in the ER, ERAP1, trims precursors to MHC class I–presented peptides

Nature Immunology volume 3, pages 1169–1176 (2002)Cite this article

Abstract

Precursors to major histocompatibility complex (MHC) class I–presented peptides with extra NH2-terminal residues can be efficiently trimmed to mature epitopes in the endoplasmic reticulum (ER). Here, we purified from liver microsomes a lumenal, soluble aminopeptidase that removes NH2-terminal residues from many antigenic precursors. It was identified as a metallopeptidase named “adipocyte-derived leucine” or “puromycin-insensitive leucine-specific” aminopeptidase. However, because we localized it to the ER, we propose it be renamed ER–aminopeptidase 1 (ERAP1). ERAP1 is inhibited by agents that block precursor trimming in ER vesicles and although it trimmed NH2-extended precursors, it spared presented peptides of 8 amino acid and less. Like other proteins involved in antigen presentation, ERAP1 is induced by interferon-γ. When overexpressed in vivo, we found that ERAP1 stimulates the processing and presentation of an antigenic precursor in the ER.

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Figure 1: Aminopeptidase activities are associated with the ER-lumenal fraction.
Figure 2: Purification and identification of ERAP1.
Figure 3: ERAP1 is localized in the ER of HeLa cells.
Figure 4: Trimming of NH2-extended precursors but not short peptides by ERAP1.
Figure 5: Immunodepletion of ERAP1 from ER lumen reduces trimming of NH2-extended precursors.
Figure 6: Effect of IFN-γ on ERAP1 expression in cultured cells.
Figure 7: IFN-γ enhances peptide trimming in ER lumen by induction of ERAP1.
Figure 8: Overexpression of ERAP1 enhances SIINFEKL generation from NH2-extended precursors in the ER.

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References

  1. Rock, K.L. et al. Inhibitors of the proteasome block the degradation of most cell proteins and the generation of peptides presented on MHC class I molecules. Cell 78, 761–771 (1994).

    Article  CAS  Google Scholar 

  2. Harding, C.V. et al. Novel dipeptide aldehydes are proteasome inhibitors and block the MHC-I antigen-processing pathway. J. Immunol. 155, 1767–1775 (1995).

    CAS  PubMed  Google Scholar 

  3. Cerundolo, V. et al. The proteasome-specific inhibitor lactacystin blocks presentation of cytotoxic T lymphocyte epitopes in human and murine cells. Eur. J. Immunol. 27, 336–341 (1997).

    Article  CAS  Google Scholar 

  4. Craiu, A. et al. Lactacystin and clasto-lactacystin β-lactone modify multiple proteasome β-subunits and inhibit intracellular protein degradation and major histocompatibility complex class I antigen presentation. J. Biol. Chem. 272, 13437–13445 (1997).

    Article  CAS  Google Scholar 

  5. Kisselev, A.F., Akopian, T.N., Woo, K.M. & Goldberg, A.L. The sizes of peptides generated from protein by mammalian 26 and 20S proteasomes. Implications for understanding the degradative mechanism and antigen presentation. J. Biol. Chem. 274, 3363–3371 (1999).

    Article  CAS  Google Scholar 

  6. Goldberg, A.L., Cascio, P., Saric, T. & Rock, K.L. The importance of the proteasome and subsequent proteolytic steps in the generation of antigenic peptides. Mol. Immunol. 39, 147–164 (2002).

    Article  CAS  Google Scholar 

  7. Botbol, V. & Scornik, O.A. Peptide intermediates in the degradation of cellular proteins. Progress Biol. Res. 180, 573–583 (1985).

    CAS  Google Scholar 

  8. Rock, K.L. & Goldberg, A.L. Degradation of cell proteins and the generation of MHC class I-presented peptides. Annu. Rev. Immunol. 17, 739–779 (1999).

    Article  CAS  Google Scholar 

  9. 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 

  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. Lucchiari-Hartz, M. et al. Cytotoxic T lymphocyte epitopes of HIV-1 Nef: Generation of multiple definitive major histocompatibility complex class I ligands by proteasomes. J. Exp. Med. 191, 239–252 (2000).

    Article  CAS  Google Scholar 

  12. Cascio, P., Hilton, C., Kisselev, A.F., Rock, K.L. & Goldberg, A.L. 26S proteasomes and immunoproteasomes produce mainly NH2-extended versions of an antigenic peptide. EMBO J. 20, 2357–2366 (2001).

    Article  CAS  Google Scholar 

  13. Paz, P., Brouwenstijn, N., Perry, R. & Shastri, N. Discrete proteolytic intermediates in the MHC class I antigen processing pathway and MHC I-dependent peptide trimming in the ER. Immunity 11, 241–251 (1999).

    Article  CAS  Google Scholar 

  14. Serwold, T., Gaw, S. & Shastri, N. ER aminopeptidase generate a unique pool of peptides for MHC class I molecules. Nature Immunol. 2, 644–651 (2001).

    Article  CAS  Google Scholar 

  15. Knuehl, C. et al. The murine cytomegalovirus pp89 immunodominant H-2Ld epitope is generated and translocated into the endoplasmic reticulum as an 11-mer precursor peptide. J. Immunol. 167, 1515–1521 (2001).

    Article  CAS  Google Scholar 

  16. Beninga, J., Rock, K.L. & Goldberg, A.L. Interferon-γ can stimulate post-proteasomal trimming of the N terminus of an antigenic peptide by inducing leucine aminopeptidase. J. Biol. Chem. 273, 18734–18742 (1998).

    Article  CAS  Google Scholar 

  17. Stoltze, L. et al. Two new proteases in the MHC class I processing pathway. Nature Immunol. 1, 413–418 (2000).

    Article  CAS  Google Scholar 

  18. Harris, C.A., Hunte, B., Krauss, M.R., Taylor, A. & Epstein, L.B. Induction of leucine aminopeptidase by interferon-γ. Identification by protein microsequencing after purification by preparative two-dimensional gel electrophoresis. J. Biol. Chem. 267, 6865–6869 (1992).

    CAS  PubMed  Google Scholar 

  19. Gaczynska, M., Rock, K.L. & Goldberg, A.L. γ-interferon and expression of MHC genes regulate peptide hydrolysis by proteasomes. Nature 365, 264–267 (1993).

    Article  CAS  Google Scholar 

  20. Gaczynska, M., Rock, K.L., Spies, T. & Goldberg, A.L. Peptidase activities of proteasomes are differentially regulated by the major histocompatibility complex-encoded genes for LMP2 and LMP7. Proc. Natl. Acad. Sci. USA 91, 9213–9217 (1994).

    Article  CAS  Google Scholar 

  21. Gaczynska, M., Goldberg, A.L., Tanaka, K., Hendil, K.B. & Rock, K.L. Proteasome subunits X and Y alter peptidase activities in opposite ways to the interferon-γ-induced subunits LMP2 and LMP7. J. Biol. Chem. 271, 17275–17280 (1996).

    Article  CAS  Google Scholar 

  22. Lauvau, G. et al. Human transporters associated with antigen processing (TAPs) select epitope precursor peptides for processing in the endoplasmic reticulum and presentation to T cells. J. Exp. Med. 190, 1227–1239 (1999).

    Article  CAS  Google Scholar 

  23. 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 

  24. 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 

  25. Elliott, T., Willis, A., Cerundolo, V. & Townsend, A. Processing of major histocompatibility class I-restricted antigens in the endoplasmic reticulum. J. Exp. Med. 181, 1481–1491 (1995).

    Article  CAS  Google Scholar 

  26. Lobigs, M., Chelvanayagam, G. & Mullbacher, A. Proteolytic processing of peptides in the lumen of the endoplasmic reticulum for antigen presentation by major histocompatibility class I. Eur. J. Immunol. 30, 1496–1506 (2000).

    Article  CAS  Google Scholar 

  27. Snyder, H.L., Yewdell, J.W. & Bennink, J.R. Trimming of antigenic peptides in an early secretory compartment. J. Exp. Med. 180, 2389–2394 (1994).

    Article  CAS  Google Scholar 

  28. Fruci, D., Niedermann, G., Butler, R.H. & van Endert, P.M. Efficient MHC class I-independent amino-terminal trimming of epitope precursor peptides in the endoplasmic reticulum. Immunity 15, 467–476 (2001).

    Article  CAS  Google Scholar 

  29. Brouwenstijn, N., Serwold, T. & Shastri, N. MHC class I molecules can direct proteolytic cleavage of antigenic precursors in the endoplasmic reticulum. Immunity 15, 95–104 (2001).

    Article  CAS  Google Scholar 

  30. Komlosh, A. et al. A role for a novel luminal endoplasmic reticulum aminopeptidase in final trimming of 26S proteasome-generated major histocompatability complex class I antigenic peptides. J. Biol. Chem. 276, 30050–30056 (2001).

    Article  CAS  Google Scholar 

  31. Lyko, F., Martoglio, B., Jungnickel, B., Rapoport, T.A. & Dobberstein, B. Signal sequence processing in rough microsomes. J. Biol. Chem. 270, 19873–19878 (1995).

    Article  CAS  Google Scholar 

  32. Snyder, H.L., Bacik, I., Yewdell, J.W., Behrens, T.W. & Bennink, J.R. Promiscuous liberation of MHC-class I-binding peptides from the C termini of membrane and soluble proteins in the secretory pathway. Eur. J. Immunol. 28, 1339–1346 (1998).

    Article  CAS  Google Scholar 

  33. Falk, K., Rötzschke, O. & Rammensee, H.-J. Cellular peptide composition governed by major histocompatibility complex class I molecules. Nature 348, 248–250 (1990).

    Article  CAS  Google Scholar 

  34. Menoret, A., Li, Z.H., Niswonger, M.L., Altmeyer, A. & Srivastava, P.K. An endoplasmic reticulum protein implicated in chaperoning peptides to major histocompatibility of class I is an aminopeptidase. J. Biol. Chem. 276, 33313–33318 (2001).

    Article  CAS  Google Scholar 

  35. Reed, R.C., Zheng, T. & Nicchitta, C.V. Grp94-associated enzymatic activities: Resolution by chromatographic fractionation. J. Biol. Chem. 277, 25082–25089 (2002).

    Article  CAS  Google Scholar 

  36. Hattori, A., Matsumoto, H., Mizutani, S. & Tsujimoto, M. Molecular cloning of adipocyte-derived leucine aminopeptidase highly related to placental leucine aminopeptidase/oxytocinase. J. Biochem. (Tokyo) 125, 931–938 (1999).

    Article  CAS  Google Scholar 

  37. Hattori, A. et al. Characterization of recombinant human adipocyte-derived leucine aminopeptidase expressed in Chinese hamster ovary cells. J. Biochem. (Tokyo) 128, 755–762 (2000).

    Article  CAS  Google Scholar 

  38. Hattori, A., Matsumoto, K., Mizutani, S. & Tsujimoto, M. Genomic organization of the human adipocyte-derived leucine aminopeptidase gene and its relationship to the placental leucine aminopeptidase/oxytocinase gene. J. Biochem. (Tokyo) 130, 235–241 (2001).

    Article  CAS  Google Scholar 

  39. Schomburg, L., Kollmus, H., Friedrichsen, S. & Bauer, K. Molecular characterization of a puromycin-insensitive leucyl-specific aminopeptidase, PILS-AP. Eur. J. Biochem. 267, 3198–3207 (2000).

    Article  CAS  Google Scholar 

  40. Roelse, J., Gromme, M., Momburg, F., Hämmerling, G. & Neefjes, J. Trimming of TAP-translocated peptides in the endoplasmic reticulum and in the cytosol during recycling. J. Exp. Med. 11, 1591–1597 (1994).

    Article  Google Scholar 

  41. Keller, S.R., Scott, H.M., Mastick, C.C., Aebersold, R. & Lienhard, G.E. Cloning and characterization of a novel insulin-regulated membrane aminopeptidase from Glut4 vesicles. J. Biol. Chem. 270, 23612–23618 (1995).

    Article  CAS  Google Scholar 

  42. Rogi, T., Tsujimoto, M., Nakazato, H., Mizutani, S. & Tomoda, Y. Human placental leucine aminopeptidase/oxytocinase. A new member of type II membrane-spanning zinc metallopeptidase family. J. Biol. Chem. 271, 56–61 (1996).

    Article  CAS  Google Scholar 

  43. Yamamoto, N. et al. Identification of 33 polymorphisms in the adipocyte-derived leucine aminopeptidase (ALAP) gene and possible association with hypertension. Hum. Mutat. 19, 251–257 (2002).

    Article  CAS  Google Scholar 

  44. Miyashita, H. et al. A mouse orthologue of puromycin-insensitive leucyl-specific aminopeptidase is expressed in endothelial cells and plays an important role in angiogenesis. Blood 99, 3241–3249 (2002).

    Article  CAS  Google Scholar 

  45. York, I.A. et al. The ER aminopeptidase, ERAP1, enhances or limits antigen presentation by trimming epitopes to 8–9 residues. Nature Immunol. 3, 1177–1184 (2002).

    Article  CAS  Google Scholar 

  46. Kohler, A. et al. The axial channel of the proteasome core particle is gated by the Rpt2 ATPase and controls both substrate entry and product release. Mol. Cell 7, 1143–1152 (2001).

    Article  CAS  Google Scholar 

  47. Whitby, F.G. et al. Structural basis for the activation of 20S proteasomes by 11S regulators. Nature 408, 115–120 (2000).

    Article  CAS  Google Scholar 

  48. Cascio, P., Call, M., Petre, B.M., Walz, T. & Goldberg, A.L. Properties of the hybrid form of the 26S proteasome containing both 19S and PA28 complexes. EMBO J. 21, 2636–2645 (2002).

    Article  CAS  Google Scholar 

  49. Walter, P. & Blobel, G. Preparation of microsomal membranes for cotranslational protein translocation. Meth. Enzymol. 96, 84–93 (1983).

    Article  CAS  Google Scholar 

  50. Wang, L. & Dobberstein, B. Oligomeric complexes involved in translocation of proteins across the membrane of the endoplasmic reticulum. FEBS Lett. 457, 316–322 (1999).

    Article  CAS  Google Scholar 

  51. Porgador, A., Yewdell, J.W., Deng, Y., Bennink, J.R. & Germain, R.N. Localization, quantitation, and in situ detection of specific peptide-MHC class I complexes using a monoclonal antibody. Immunity 6, 715–726 (1997).

    Article  CAS  Google Scholar 

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Acknowledgements

We thank S. Trombley for assistance in preparing this manuscript, A. Tibebu Kassa in for help with experiments and M. Jedrychowski and S. Gygi for LC-MS/MS analyses. Supported by grants from the NIH (to A. L. G. and K. L. R.).

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  1. Tomo Saric

    Present address: ATABIS GmbH, Joseph-Stelzmann Str. 50, 50931, Cologne, Germany

Authors and Affiliations

  1. Department of Cell Biology, Harvard Medical School, Boston, 02115, MA, USA

    Tomo Saric, Shih-Chung Chang, Shirley Markant & Alfred L. Goldberg

  2. Laboratory of Cellular Biochemistry, RIKEN, 2-1 Hirosawa, Wako, 351-0198, Saitama, Japan

    Akira Hattori & Masafumi Tsujimoto

  3. Department of Pathology, University of Massachusetts Medical School, Worcester, 06155, MA, USA

    Ian A. York & Kenneth L. Rock

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Supplementary information

Web Fig. 1.

Gel filtration chromatography of ER-lumenal proteins. (a) The ER-lumenal proteins were fractionated on a Sephacryl S-200 HR size exclusion column. The elution positions of aldolase (158 kD), carbonic anhydrase (29 kD) and cytochrome C (12.4 kD) are marked with arrows. Only one peak of aminopeptidase activity was detected with an apparent molecular weight of ~150 kD (Kav = 0.1083). (b) Fractions encompassing the aminopeptidase peak (23–36) were analyzed by electrophoresis on 4–12% NuPAGE gel and the elution profiles of ERAP1 (A-LAP) and Grp94 (gp96) were determined by immunoblotting. (PDF 508 kb)

Web Fig. 2.

Recombinant ERAP1 can remove a variety of residues from NH2-extended antigenic peptides. (a) Reactions contained 150 nmol/ml of QLESIINFEKL and 3 μg/ml of recombinant human enzyme. At the indicated times, an aliquot was removed and fractionated by RP-HPLC. (b) Reactions with indicated peptides were performed and analyzed as in a. The amount of a peptide trimmed was calculated by integration of peptide peaks. (PDF 277 kb)

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Saric, T., Chang, SC., Hattori, A. et al. An IFN-γ–induced aminopeptidase in the ER, ERAP1, trims precursors to MHC class I–presented peptides. Nat Immunol 3, 1169–1176 (2002). https://doi.org/10.1038/ni859

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