Concerted peptide trimming by human ERAP1 and ERAP2 aminopeptidase complexes in the endoplasmic reticulum

© 2005 Nature Publishing Group http://www.nature.com/natureimmunology ARTICLES Concerted peptide trimming by human ERAP1 and ERAP2 aminopeptidase complexes in the endoplasmic reticulum Loredana Saveanu1, Oliver Carroll1, Vivian Lindo2, Margarita Del Val3, Daniel Lopez3, Yves Lepelletier4, Fiona Greer2, Lutz Schomburg5, Doriana Fruci1,7, Gabriele Niedermann6,7 & Peter M van Endert1 The generation of many HLA class I peptides entails a final trimming step in the endoplasmic reticulum that, in humans, is accomplished by two ‘candidate’ aminopeptidases. We show here that one of these, ERAP1, was unable to remove several N-terminal amino acids that were trimmed efficiently by the second enzyme, ERAP2. This trimming of a longer peptide required the concerted action of both ERAP1 and ERAP2, both for in vitro digestion and in vivo for cellular antigen presentation. ERAP1 and ERAP2 localized together in vivo and associated physically in complexes that were most likely heterodimeric. Thus, the human endoplasmic reticulum is equipped with a pair of trimming aminopeptidases that have complementary functions in HLA class I peptide presentation. Major histocompatibility complex (MHC) class I molecules present degradation products of cellular proteins that are supplied by the antigen-processing machinery of the presenting cell. This machinery ‘co-opts’ cellular proteolytic systems for producing peptides for MHC class I molecules1. The initial degradation of antigenic proteins is accomplished by cytosolic proteasome complexes, whose cleavages create the C-terminal ends of peptides presented by MHC class I because of the absence of carboxypeptidase activities in the MHC class I processing pathway2. In contrast, aminopeptidases with the potential to trim precursors of MHC class I peptides have been identified both in the cytosol and in the endoplasmic reticulum (ER)1. In the cytosol, proteasome products relevant for antigen presentation may often have a length greater than 15 residues and require trimming by tripeptidyl peptidase II to render them suitable for further handling by the processing system3. Enzymes acting ‘downstream’ presumably increase the variety of peptides available for MHC class I presentation and/or help to adapt them to the binding requirements of MHC class I molecules. ER trimming enzymes are also essential when final peptides presented by HLA class I are poorly translocated into the ER; this may occur often for some HLA class I ‘allomorphs’ that bind peptides with anchor positions conferring low affinity for transporter associated with antigen processing (TAP)4,5. In such cases, transport of precursor peptides with higher affinities for TAP, followed by Nterminal trimming by luminal peptidases, is required for efficient peptide presentation4,6. Considerable progress has been made in the elucidation of peptide trimming in the ER. A metallopeptidase that has been described in other contexts with various designations7–9 has been proposed to represent the principal ER enzyme for the trimming of MHC class I ligand precursors10–12. Mouse ER aminopeptidase associated with antigen processing (ERAAP), also known as ER aminopeptidase 1 (ERAP1) in humans, localizes together with ER markers, is induced by interferon-g (IFN-g) and shows an unusual preference for peptides with a length of nine or more residues. Experiments using RNA interference have suggested that ERAP1 may be involved in the formation of about one third of peptide–MHC class I complexes10,11. Nevertheless, the net effect of ERAP1 on antigen presentation reportedly varies according to the cell type or epitope studied. ‘Knockdown’ of ERAP1 mRNA in untreated HeLa cells increases cell surface HLA class I expression, whereas the same treatment of IFN-g-induced cells reduces it10,11. Given that the enzyme has been reported to trim peptides with little sequence specificity, other than poor cleavage of the X-Pro bond (where ‘X’ is any amino acid), the reasons for these phenomena remain unclear12,13. A second putative human trimming peptidase has been identified based on its homology with ERAP1 (ref. 14). Like ERAP1, this leukocyte-derived arginine aminopeptidase (L-RAP) is localized in the ER and is induced by IFN-g. In vitro trimming of several synthetic precursor peptides by recombinant L-RAP suggested that the enzyme might also be involved in precursor trimming in the ER14. The identification of L-RAP 1Institut National de la Sante et Recherche Médicale Unité 580; Université René Descartes Paris 5, 75015 Paris, France. 2M-SCAN, Fishponds Close, Wokingham, Berkshire RG41 2TZ, UK. 3Centro Nacional de Microbiologı́a, Instituto de Salud Carlos III, 28220 Majadahonda, Spain. 4Centre National de la Recherche Scientifique UMR 8147, Necker Institute, 75015 Paris, France. 5Institut für Experimentelle Endokrinologie, Charité-Universitätsmedizin Berlin, 10117 Berlin, Germany. 6Max-Planck Institut für Immunbiologie, 79108 Freiburg, Germany. 7Present addresses: Research Center Ospedale Bambino Gesù, 00165 Rome, Italy (D.F.) and Clinic of Radiation Oncology, Experimental Division, University Clinic, Freiburg, Germany (G.N.). Correspondence should be addressed to P.M.v.E. (). Published online 22 May 2005; doi:10.1038/ni1208 NATURE IMMUNOLOGY VOLUME 6 NUMBER 7 JULY 2005 689 20 b 10 Leu-AMC 5 0 1 10 20 30 40 50 60 70 80 90 100 20 Fluorescence units (×103) © 2005 Nature Publishing Group http://www.nature.com/natureimmunology + CHAPS – CHAPS 15 S9L R10L a Fluorescence units (×103) ARTICLES –CHAPS 7 18 19 23 25 + CHAPS 30 39 40 52 80 84 c 15 Blot: ERAP 1 ERAP 2 10 Arg-AMC 5 0 1 10 20 30 40 50 60 70 Fraction Peak 80 90 7 19 23 1 25 2 30 39 84 3 4 7 19 23 1 25 30 39 84 2 3 4 100 Fraction number Figure 1 Multiple aminopeptidase activities are associated with human B cell microsomes. (a) Aminopeptidase activity of fractionated ER samples. Microsomes prepared from 2
109 MGAR B cells were digested with proteinase K to remove extraluminal peptidases, were lysed in PBS with 1% CHAPS and were injected onto an anion-exchange column. Proteins were eluted sequentially by NaCl gradients in the absence ( CHAPS) and presence (+ CHAPS) of 0.5% CHAPS. A portion (150 ml) of each fraction was tested for activity against Leu-AMC substrate (top) or Arg-AMC substrate (bottom). (b) TLC analysis of 7.5 ml of the fractions obtained in a (fractions, below lanes), after incubation for 10 min with 0.5 ml of 125I-labeled R10L. Far left, undigested labeled reference peptides S9L and R10L. (c) Immunoblot analysis of 10% of the fractions obtained in a (fractions, below lanes). Samples were concentrated and were analyzed by immunoblot with mAbs specific for ERAP1 (6H9) or ERAP2 (3F5). Data are representative of at least ten experiments (a,b) or of two experiments (c). raised the question of why the human ER is equipped with two trimming aminopeptidases. An ER activity has been descri (...truncated)