www.nature.com/natureimmunology • december 2002 • volume 3 no 12 • nature immunology Tomo Saric 1 *, Shih-Chung Chang 1 , Akira Hattori 2 , Ian A.York 3 , Shirley Markant 1 , Kenneth L. Rock 3 , Masafumi Tsujimoto 2 and Alfred L. Goldberg 1 Published online 18 November 2002; doi:10.1038/ni859 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 NH 2 - 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 NH 2 - 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. 1 Department of Cell Biology, Harvard Medical School, Boston, MA 02115, USA. 2 Laboratory of Cellular Biochemistry, RIKEN, 2-1 Hirosawa,Wako, Saitama, 351-0198 Japan. 3 Department of Pathology, University of Massachusetts Medical School,Worcester, MA 06155, USA. *Present address:ATABIS GmbH, Joseph-Stelzmann Str. 50, 50931 Cologne, Germany. Correspondence should be addressed to A. L. G. (alfred_goldberg@hms.harvard.edu). An IFN-γ–induced aminopeptidase in the ER, ERAP1, trims precursors to MHC class I–presented peptides The ability of the immune system to recognize and eliminate virally infected and cancer cells depends upon the cells’ capacity to present on its surface major histocompatibility complex (MHC) class I molecules peptide fragments derived from intracellular proteins. Most of these anti- genic peptides are derived from peptides generated in the cytosol or nucleus during protein degradation by the ubiquitin-proteasome path- way 1–4 . The 26S proteasome degrades proteins to peptides of ∼2–25 residues long 5,6 . Nearly all these peptides are hydrolyzed quickly to amino acids by cytosolic peptidases 6,7 . However, in higher vertebrates, a small fraction escapes complete degradation and is transported into the endoplasmic reticulum (ER), where it binds to MHC class I molecules and is exported to the cell surface 8 . To fit into the groove in MHC class I molecules, these peptides must be 8–11 residues long. Approximately 70% of proteasome products are too short to do so 5 , about 15% are of appropriate length and 15% are too long, but could function in antigen presentation if trimmed by exopeptidases 5,6,12 . Studies with inhibitors have shown that proteasomes generate the correct COOH terminus for the MHC class I–presented peptide 9,10 and sometimes also the correct NH2 terminus 11,12 . However, proteasomes—and especially the alternative forms induced by interferon-γ (IFN-γ29 termed “immunopro- teasomes” 12 —primarily generate longer precursors of MHC class I epi- topes that are extended at their NH2 termini by one or more residues 12–15 . The conversion of these NH2-extended precursors to mature epitopes is not catalyzed by proteasomes 9 but by aminopeptidases, as this process can be blocked by derivatization of the peptide’s NH2-terminal group 10 . This trimming of precursor peptides to mature epitopes can occur in the cytosol or the ER. Three aminopeptidases have been implicated in this cytosolic process: leucine aminopeptidase (LAP) 16 , puromycin-sensitive aminopeptidase and bleomycin hydrolase 17 . One of these cytosolic enzymes, LAP, is induced by IFN-γ 16 , which stimulates antigen presenta- tion, and thus LAP is likely to play a special role in processing antigenic precursors in vivo. IFN-γ also stimulates the induction of other compo- nents of this pathway; these components include MHC class I, the peptide transporter (TAP) and three proteasome β-subunits, whose incorporation in place of the constitutive subunits alters the cleavage specificity so that more peptides are generated with the correct COOH-terminal residues for MHC class I binding 12,19–21 . In addition, these changes in specificity influ- ence the NH2 termini of the proteasomal products and increase specifical- ly the yield of NH2-extended precursors to antigenic peptides 12 . Aminopeptidase(s) in the ER also can contribute to the generation of mature MHC class I epitopes from these larger precursors. In fact, cer- tain MHC class I epitopes appear to be transported into the ER primar- ily as NH2-extended peptides. This is probably because such precursors have higher affinities for TAP than the mature epitopes 22,23 and perhaps because these longer forms are destroyed more slowly by peptidases in the cytosol 6,24 . A variety of studies have demonstrated proteolytic pro- cessing of precursor peptides transported into the ER in vivo and in iso- lated microsomes. For example, when NH2-extended versions of anti- genic peptides fused to an ER-targeting sequence were expressed in cells, the mature epitopes were presented on surface MHC class I mol- ecules 9,25–27 after cleavage of the signal sequences by signal peptidase and removal of the additional NH2-terminal residues by aminopepti- dase(s) in the ER. Studies with purified microsomes have demonstrated that this trimming of TAP-translocated precursors occurs in the lumen of A RTICLES 1169 © 2002 Nature Publishing Group http://www.nature.com/natureimmunology