Cytosol Is Mediated by Two Peptidases Class I-Restricted

The Final N-Terminal Trimming of a Subaminoterminal Proline-Containing HLA Class I-Restricted Antigenic Peptide in the Cytosol Is Mediated by Two Peptidases This information is current as of September 23, 2021. Frédéric Lévy, Lena Burri, Sandra Morel, Anne-Lise Peitrequin, Nicole Lévy, Angela Bachi, Ulf Hellman, Benoît J. Van den Eynde and Catherine Servis J Immunol 2002; 169:4161-4171; ;

doi: 10.4049/jimmunol.169.8.4161 Downloaded from http://www.jimmunol.org/content/169/8/4161

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2002 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

The Final N-Terminal Trimming of a Subaminoterminal Proline-Containing HLA Class I-Restricted Antigenic Peptide in the Cytosol Is Mediated by Two Peptidases1

Fre´de´ric Le´vy,2,3* Lena Burri,2* Sandra Morel,4† Anne-Lise Peitrequin,* Nicole Le´vy,‡ Angela Bachi,§ Ulf Hellman,¶ Benoıˆt J. Van den Eynde,† and Catherine Servis‡

The proteasome produces MHC class I-restricted antigenic peptides carrying N-terminal extensions, which are trimmed by other peptidases in the cytosol or within the endoplasmic reticulum. In this study, we show that the N-terminal editing of an antigenic peptide with a predicted low TAP affinity can occur in the cytosol. Using proteomics, we identified two cytosolic peptidases, tripeptidyl peptidase II and puromycin-sensitive aminopeptidase, that trimmed the N-terminal extensions of the precursors pro- Downloaded from duced by the proteasome, and led to a transient enrichment of the final antigenic peptide. These peptidases acted either sequentially or redundantly, depending on the extension remaining at the N terminus of the peptides released from the proteasome. Inhibition of these peptidases abolished the CTL-mediated recognition of Ag-expressing cells. Although we observed some proteolytic activity in fractions enriched in endoplasmic reticulum, it could not compensate for the loss of tripeptidyl peptidase II/puromycin-sensitive aminopeptidase activities. The Journal of Immunology, 2002, 169: 4161–4171. http://www.jimmunol.org/ igands of the MHC class I molecules are composed of that other peptidases, in the endoplasmic reticulum (ER)5 or in the peptides of 8–10 aa in length. Normally, these peptides cytosol, are involved in the N-terminal trimming of precursor pep- L are derived from the pool of intracellular polypeptides tides (5). Although ER resident proteases have been inferred to translated in the various cell types. During viral infection or in function in the Ag-processing pathway, none have yet been de- tumor cells, the pool of MHC class I-restricted peptides also in- scribed at the molecular level (6–12). In contrast, several cytosolic cludes those derived from proteins encoded by virus or tumor- peptidases potentially involved in this process have been identi- specific genes. Those peptides represent a crucial component of the fied. Those include puromycin-sensitive aminopeptidase, bleomy- specific recognition and lysis of the abnormal cells by CTL. cin hydrolase, thimet oligopeptidase, and leucyl aminopeptidase An essential step in the production of the MHC class I-restricted (13–15). A common feature of these peptidases is that they display by guest on September 23, 2021 peptides is the degradation of proteins by the proteasome (1). The a broad specificity and do not lead to an enrichment of the exact proteasome is a large multicatalytic protease, present in the cytosol antigenic peptide. In some instances, the generation of the accurate N terminus of antigenic peptides can be mediated by more than and the nucleus of eukaryotic cells, which degrades the bulk of one peptidase in a redundant manner (13). intracellular proteins and generates peptides ranging from 3 to 22 The human gene RU1 codes for a ubiquitously expressed intra- aa in length (2). Whereas some antigenic peptides are directly pro- cellular protein of unknown function. A CTL clone, originally duced by the proteasome in their final form, others are produced as raised against an autologous renal carcinoma, recognized an HLA- precursor peptides (3, 4). Those precursor peptides display the ex- B51-restricted peptide derived from the RU1 region spanning aa act C terminus of the final antigenic peptides, but carry N-terminal 34–42 (RU134–42), with sequence VPYGSFKHV (16). In accor- amino acid extensions of various lengths. It is therefore assumed dance with the predicted HLA-B51-specific anchor motifs, this peptide contained a proline (P) at position 2 and valine (V) at position 9 (17). In vitro studies using precursor peptides encompassing the antigenic region demonstrated that the exact C termi- *Ludwig Institute for Cancer Research, Lausanne Branch, and †Institute of Biochem- nus of this CTL-defined epitope was directly produced by the stan- istry, University of Lausanne, Epalinges, Switzerland; ‡Ludwig Institute for Cancer Research, Brussels Branch, Universite´ Catholique de Louvain, Brussels, Belgium; dard proteasome, but not the final N terminus. Consequently, other §DIBIT, San Raffaele Scientific Institute, Milan, Italy; and ¶Ludwig Institute for peptidases could act on the N-terminal extension to produce the Cancer Research, Uppsala Branch, Biomedical Center, Uppsala, Sweden antigenic nonamer. Based on several in vitro studies indicating that Received for publication May 1, 2002. Accepted for publication August 7, 2002. antigenic peptides containing a subaminoterminal P are poor sub- The costs of publication of this article were defrayed in part by the payment of page strates for the human TAP transporters (7, 18–20), it has been charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. inferred that such peptides are transported through the TAP as 1 This work was supported in part by a grant of the Swiss National Fund (to F.L.), by an Investigator Award of the Cancer Research Institute (to F.L.), and by a grant of the Swiss Cancer League (to L.B.). 2 F.L. and L.B. contributed equally to this study. 5 Abbreviations used in this paper: ER, endoplasmic reticulum; AMC, 7-amido-4- methylcoumarin; CMK, chloromethylketone; 2-D, two-dimensional; EBNA, EBV- 3 Address correspondence and reprint requests to Dr. Fre´de´ric Le´vy, Ludwig Institute encoded nuclear Ag; EGFP, enhanced GFP; GFP, green fluorescent protein; HA, for Cancer Research, Ch. des Boveresses 155, CH-1066 Epalinges, Switzerland. hemagglutinin; MALDI-TOF, matrix-assisted laser desorption ionization-time of E-mail address: [email protected] flight; MS, mass spectrometry; PSA, puromycin-sensitive aminopeptidase; RCC, re- 4 Current address: Institute of Experimental Immunology, Department of Pathology, nal cell carcinoma; RT, room temperature; TFA, trifluoroacetic acid; TPP II, tripep- University of Zu¨rich, CH-8091 Zurich, Switzerland. tidyl peptidase II; Ub, ubiquitin; VSV, vesicular stomatitis virus.

N-terminal extended precursors and that the final N terminus is TPP II was purified from 5 ϫ 107 HEK293 cells by affinity purification, generated by peptidases localized within the ER (7, 21). using 5 ␮l polyclonal chicken Ab anti-human TPP II (Immunsystem, Upp- ␮ Contrary to this prediction, our results indicate that the antigenic sala, Sweden), 5 g anti-chicken biotin conjugate Ab (Promega, Madison, WI), and 20 ␮l streptavidin-coated agarose beads (Pierce, Rockford, IL). peptide RU134–42, which contains P at second position, can be The plasmid PSA-vesicular stomatitis virus (VSV), directing the synthesis produced in the cytosol, before TAP-mediated transport. By using of PSA carrying a C-terminal Ab epitope from the vesicular stomatitis a substrate-based assay, we identified two cytosolic peptidases that virus (a generous gift of A. Fontana, University Hospital, Zu¨rich, Switzer- 7 trim the N terminus of the RU1 precursors produced by the land), was transiently transfected into 4 ϫ 10 HEK293 cells using Fugene 34–42 (Roche, Basel, Switzerland) and the manufacturer-supplied protocol. proteasome. These two peptidases, tripeptidyl peptidase II (TPP II) Twenty-four hours posttransfection, cells were lysed in 1% Triton X-100, and puromycin-sensitive aminopeptidase (PSA), act on the N-ter- and PSA-VSV was immunoprecipitated using 5 ␮g anti-VSV tag mAb minally extended precursors to produce, and transiently enrich for, (Fluka, Buchs, Switzerland) and 20 ␮l protein G-Sepharose slurry (Pierce). the exact N terminus of the antigenic peptide. We also searched for Due to the limited number of cells used in this assay, the final purity of the proteolytic activities in membranes enriched in ER. Although we isolated peptidases could not be ascertained. However, the specificities of both Abs have been described by others (23, 24), and the proteolytic ac- observed a detectable proteolytic activity against one of the pro- tivities of the TPP II and PSA preparations could be completely blocked by teasomal products, RU131–42, it could apparently not rescue the butabindide and puromycin, respectively (data not shown). The immuno- loss of CTL recognition of tumor cells resulting from the inhibition precipitated material was washed four times and was used to digest 4 nmol of proteasome and TPP II/PSA activities. Our data suggest that the peptides of interest. At the end of the digestion, the supernatant was col- lected and the digestion products were analyzed by MS. production of RU134–42/HLA-B51 is a cytosolic process, involving the proteasome to generate the exact C terminus of the anti- Preparative 2-D gel electrophoresis genic peptide and TPP II and PSA to trim the N-terminal exten- Downloaded from The protein pellets obtained from the above-mentioned fractions 10–14 sions produced by the proteasome. were resuspended in 600 ␮l loading buffer (40 mM Tris-HCl, pH 8.0, 8 M urea, 4% CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propane- Materials and Methods sulfonate), 65 mM dithioerythritol, 0.01% bromphenol blue), loaded onto 18-cm-long nonlinear, pH 3–10 gradient strips (Amersham Pharmacia Bio- Protein purification tech, Piscataway, NJ), and separated overnight by electrophoresis. During ϫ the initial 3 h, the voltage was linearly increased from 300 to 3500 V, For the identification of TPP II, BB64-renal cell carcinoma (RCC) (6.3 http://www.jimmunol.org/ 108 cells) were mechanically disrupted by douncing in a Dounce homo- followed by3hat3500 V, to reach the final voltage of 5000 V. After genator. Sucrose was immediately added to the homogenate to a final con- separation in the first dimension, the strips were equilibrated in 50 mM centration of 250 mM. Debris were removed by centrifugation at 13,000 ϫ Tris-HCl, pH 8.4, 6 M urea, 30% glycerol, 2% SDS, and 2% dithioeryth- g for 15 min at 4°C. The supernatant was transferred to ultracentrifuge ritol for 12 min. Thiol groups were subsequently blocked with 2.5% io- tubes and subjected to ultracentrifugation at 80,000 ϫ g for 45 min at 4°C. doacetamide. Separation in the second dimension was conducted using a Proteasomes were removed from the clear supernatant (complete cytosol) vertical gradient slab gel with a modified Laemmli-SDS discontinuous sys- ϳ by affinity purification, using the mAb anti-proteasome MCP21 (22). Pro- tem (10% acrylamide-piperazine diacrylyl gel) and run at 200 V for 5h. teasome-depleted cytosol was loaded onto a high performance ion-ex- Gels were silver stained according to a protocol compatible with MS (25). change Source 15Q PE 4.6/100 Sepharose column (Amersham Pharmacia Biotech, Piscataway, NJ) at a flow rate of 1 ml/min in buffer A (20 mM Destaining, in-gel protein digestion, extraction, and purification Tris-HCl, pH 7.6), washed with five-column volumes of buffer A, and Each visible spot of the 2-D gel was cut and lyophilized in a sterile Ep- by guest on September 23, 2021 pre-eluted with 30% buffer B (20 mM Tris-HCl, pH 7.6, 1 M NaCl). The pendorf tube. The silver stain was removed by covering the gel piece with adsorbed material was then eluted, using a linear gradient of 30–70% 30 mM K-ferricyanide and 100 mM Na-thiosulfate (1:1, v/v), shaking for ␮ buffer B, in 35 fractions of 1 ml each. An aliquot of each fraction (50 l) some minutes, and observing the destaining (26). Each gel piece was then was incubated with 4 nmol peptide RU129–47 (TGSTAVPYGSFKH washed with water three times, covered with 0.2 M ammonium bicarbon- VDTRLQ, in one-letter code, in which the underlined sequence corre- ate, and incubated for at least 20 min at room temperature (RT). The am- sponds to the final CTL-defined antigenic peptide) for1hat37°C. The monium bicarbonate was then removed, replaced with 100% acetonitrile, reaction was stopped by adding trifluoroacetic acid (TFA) to a final con- and washed three times. Each gel piece was dried, and the digestion was centration of 2%. The samples were then analyzed by matrix-assisted laser started by adding trypsin (Promega; 0.5 ␮g/gel piece) in 0.2 M ammonium desorption ionization-time of flight (MALDI-TOF) mass spectrometry bicarbonate and kept on ice for 15–20 min. More buffer was added in small (MS), as previously described (22). The positive fractions (fractions 10– aliquots to allow a slow uptake of the protease into the gel. The digestion ␮ 14) were pooled, concentrated to a final volume of 500 l, and precipitated was conducted overnight at 37°C. The reaction was stopped by adding TFA with 20% TCA overnight at 4°C. The precipitated proteins were pelleted at to a final concentration of 1%, and the sample was sonicated for 10 min. ϫ Ϫ 13,000 g for 10 min at 4°C, washed three times with 100% cold ( 20°C) The supernatant was saved, 0.1% TFA/60% acetonitrile was added to cover acetone, and lyophilized. The proteins were then subjected to two-dimen- each gel piece, and the tube was incubated for at least 30 min at 37°C. This sional gel electrophoresis (2-D gel). extract was combined with the previous supernatant, and extraction was For the identification of PSA, proteasome-depleted cytosol from 100 ml repeated. A final extraction was performed, using 100% acetonitrile. All concentrated human erythrocytes was loaded onto a DEAE-32 column, extracts were then combined, and the volume was reduced by vacuum pre-equilibrated in 10 mM phosphate buffer, pH 7.5. The column was ex- centrifugation to about one-third of the original volume. The samples were ϫ tensively washed, and the adsorbed material was eluted in 30 1-ml desalted by passing them though Zip-Tips (C18; Millipore, Bedford, MA). fractions with 200 mM phosphate buffer, pH 7.5. The eluted material was ␮ The samples were lyophilized and resuspended in 3 l 0.1% TFA/H20. then desalted on a Sephacryl S-300 column, in 20 mM Tris-HCl, pH 7.6, One microliter of the sample was mixed (1:1, v/v) with matrix (a saturated and loaded onto a Source 15Q PE 4.6/100 column, at a flow of 1 ml/min. solution of ␣-cyano-4-hydroxycinnamic acid in 50% acetonitrile/0.1% After extensive washing with buffer A, the adsorbed material was eluted in TFA) and spotted on the target of the mass spectrometer. ϫ 60 1-ml fractions, using a linear gradient of 0–65% buffer B. An aliquot Treatment of the bands excised from the Coomassie-stained gel was ␮ ␮ (20 l) of each fraction was tested with 100 M H-Ala-Ala-Phe-amidom- identical, except that the K-ferricyanide/Na-thiosulfate treatment was ethylcoumarin (AAF-AMC) for 15 min at 37°C. Proteolytic activity was omitted. monitored by the increased fluorescence resulting from the release of the AMC group (excitation/emission 380/440 nm). A total of 4 nmol peptide Synthetic peptide digestion assays ␮ RU132–47 was incubated with 12 l fractions 29, 31, and 32, respectively, for 20 min at 37°C. The digestion was stopped with 2% TFA and analyzed A total of 4 nmol peptide was used in digestion assays with cytosol, its by MS as above. Fraction 30 was precipitated with 20% TCA and pro- derived fractions, and purified peptidases. The digestions were allowed to cessed as above. After lyophilization, the protein pellet was resuspended in proceed for the time indicated in the figures, and the reactions were stopped SDS-sample buffer containing DTT and boiled for 3 min at 95°C. After by adding 2% TFA. After lyophilization, the samples were analyzed by cooling, iodoacetamide was added to the samples before separation by MS, as previously described (22). In digestion performed in the presence of SDS-12% PAGE. The gel was stained with 0.2% Coomassie brilliant blue peptidase inhibitors, the inhibitors were added to the cytosol and incubated R in 20% methanol/0.5% acetic acid and destained with 20% methanol, for 15 min at RT prior to the addition of peptide. The concentration of and the visible bands were excised and treated, as described below. butabindide (a kind gift of J.-C. Schwartz, Institut National de la Sante´ et The Journal of Immunology 4163 de la Recherche Me´dicale Unite´ 109, Paris, France) was 5 ␮M, AAF- EGFP-Ub plasmid control coded for EGFP-Ub-Melan-AMART1, in which chloromethylketone (CMK) 50 ␮M, and puromycin 50 ␮M. Melan-AMART1 is a melanoma-associated protein of 118 aa. MALDI-TOF mass-spectroscopic analysis and database search Cell lines, transient transfections, and metabolic labeling For protein identification, all analyses were performed using a Perseptive Biosystems MALDI-TOF Voyager DE-RP or a Voyager DE-STR mass The human renal carcinoma BB64-RCC and the human embryonic kidney spectrometer (Framingham, MA) operated in the delayed extraction and HEK293 and HEK293EBNA (EBV-encoded nuclear Ag) cell lines were reflector mode. The search program ProFound, developed by The Rock- maintained in DMEM medium (Invitrogen), supplemented with 10% FCS, efeller Mass Spectrometry Laboratory and New York University (New antibiotics, and 20 mM Na HEPES, pH 7.3. York, NY), was used for database searches (27). Peptides were selected in Transient transfections of the HEK293-EBNA cells were performed us- the mass range of 800-4000 Da. Spectra were calibrated using a matrix and ing the Lipofectamine reagent (Invitrogen). Fifty thousand cells were tryptic autodigestion ion peaks as internal standards. plated into flat-bottom microwells and transfected with 1.5 ␮l Lipo- For regular peptide digestion assays, the settings of the instrument were fectamine, 20 ng plasmid pcDNA3 containing the HLA-B*5101 cDNA, as reported (22). and 12.5 ng of either pcDNA3.1TOPO plasmid (Invitrogen) containing the RU1 full-length cDNA, pEGFP/Ub plasmid containing the cDNA-encod-

Cell fractionation ing RU134–42, or pEGFP/Ub-encoding RU134–47. A total of 100 ng pBJ1neo construct encoding the herpes simplex-derived TAP inhibitor ϫ 8 BB64-RCC (2 10 cells) were mechanically disrupted in hypotonic ICP47 molecule (a kind gift of H. G. Rammensee, Tu¨bingen, Germany) buffer, and the membranes were immediately equilibrated with 250 mM was added to this mix in one-half of the wells. After 20 h, the transfected ϫ sucrose. Cell debris were pelleted at 13,000 g for 10 min. The super- cells were incubated with 104 anti-RU1 /HLA-B51 CTL 381/84, along ϫ 34–42 natant was subjected to high speed centrifugation (80,000 g) for 45 min with 25 U/ml IL-2. The amount of TNF-␣ secreted in the supernatant was at 4°C. Incomplete cytosol (6 ml) was obtained by the removal of the assessed 24 h later by ELISA (Endogen, Woburn, MA). Downloaded from proteasome from the clear supernatant, using immobilized anti-proteasome For metabolic labeling, 2 ϫ 106 HEK293 cells were transfected with 4 Ab MCP21. Membranes were resuspended in 2 ml RM buffer (50 mM ␮g plasmid using the Fugene reagent (Roche) and following the manufac- HEPES, pH 7.2, 250 mM sucrose, 50 mM potassium acetate, 2 mM mag- turer’s protocol. Sixteen hours posttransfection, the cells were starved for ϫ nesium acetate) and pelleted at 80,000 g for 45 min at 4°C. After re- 45 min in Met/Cys-free DMEM medium (ICN Biomedicals, Aurora, OH) peating this procedure twice, the membranes were resuspended in 1 ml RM at 37°C. The cells were metabolically labeled for 20 min at 37°C in fresh buffer containing 2% Triton X-114, maintained on ice for 10 min, and Met/Cys-free medium containing 150 ␮Ci [35S]Met/Cys (Pro-mix; Amer- subjected to phase separation (28). This was performed by placing the sham Pharmacia Biotech). Cells were washed once and lysed in 1 ml lysis ϫ samples at 37°C for 10 min and by subsequent centrifugation at 12,000 buffer containing 1% Triton X-100 and 30 mM iodoacetamide to prevent http://www.jimmunol.org/ g for 10 min at RT. The upper phase (fraction A), corresponding to the postlysis deubiquitylation. Unsolubilized material was removed by centrif- detergent-poor fraction and containing hydrophilic luminal proteins, was ugation, and the supernatant was incubated with saturating amounts of the transferred into a new tube, and the remaining detergent-rich lower phase mAb anti-hemagglutinin (HA) epitope (Berkeley Antibody, Richmond, (fraction B), containing membrane-anchored proteins, was washed by add- CA) and 20 ␮l protein G-Sepharose. The immunoprecipitates were washed ing fresh RM buffer lacking Triton X-114. The tube was placed on ice for and treated, as described earlier (22), subjected to SDS-12% PAGE, fol- 10 min and the phase separation was repeated. After two washing cycles, lowed by autoradiography. the volume of the detergent-rich phase was readjusted to the original volume (1 ml). Twenty microliters of incomplete cytosol, 4 ␮l fraction A, and 4 ␮l fraction B, which corresponds to the material obtained from an equiv- Treatment of target cells with inhibitors of Ag processing alent number of cells, were incubated with 4 nmol RU1 peptides for 20 min

BB64-RCC cells were acid treated as follows to discard peptides from the by guest on September 23, 2021 at 37°C. The reaction was stopped by adding 2% TFA, and the samples surface HLA molecules. One million cells were incubated for exactly 30 s were analyzed by MS, as described above. Forty microliters of incomplete at 37°Cin500␮l 300 mM glycine buffer (pH 2.5), supplemented with 1% cytosol, 8 ␮l phase A, and 8 ␮l phase B were incubated with 100 ␮M sterile BSA, and then washed several times with culture medium. Eight AAF-AMC for 10 min at 37°C. Where indicated, the peptidase inhibitors thousand cells seeded in microplates were incubated for 14 h with 50 ␮M butabindide (5 ␮M) and puromycin (50 ␮M) were added 15 min before the lactacystin (Calbiochem, La Jolla, CA) or 100 ␮M AAF-CMK in culture addition of the fluorogenic substrate. medium. Thereafter, some of the cells were pulsed for 30 min with the RU1 Western blot analysis antigenic peptide VPYGSFKHV at a final concentration of 10 ␮M, washed, and incubated with 3000 CTL 381/84 and 25 U/ml IL-2. The Western blot analysis was performed according to standard procedures. A ability of the treated cells to stimulate the CTL was assessed by measuring total of 20 ␮l cytosol, 4 ␮l fraction A, and 4 ␮l fraction B was separated the production of TNF by the CTL after 20 h of incubation. The TNF (␣ on SDS-8% PAGE for the detection of TPP II and SDS-10% PAGE for the and ␤) content of the supernatants was evaluated by testing their cytotoxic detection of PSA, and blotted onto nitrocellulose. The polyclonal chicken effect on WEHI-164 clone 13 cells (29). anti-human TPP II Ab (Immunsystem), anti-chicken biotin conjugate Ab (Promega), and the streptavidin-HRP conjugate (Invitrogen, San Diego, CA) were used at a 1/1000 dilution to reveal TPP II. For the detection of Results PSA, a goat Ab anti-PSA (1:1000) Ab and a second Ab anti-goat peroxi- TPP II mediates the initial trimming of RU129Ð47 precursors, dase conjugate (1:3000) (Fluka) were used. The protein signals were re- but fails to produce the final N terminus of the antigenic peptide vealed with ECL Western blotting detection reagent (Amersham Pharma- cia Biotech). Earlier work, using the precursor peptide RU124–47, demonstrated Cloning of RU1 minigenes that the exact C terminus of the antigenic peptide was directly produced by purified standard proteasome, but that the N terminus The pEGFP-Ub vector is similar to the one described previously (22), always carried additional 3 (RU1 ) and 5 (RU1 ) aa (16) except that the green fluorescent protein (GFP) moiety has been replaced 31–42 29–42 by enhanced GFP (EGFP). Details are available upon request. The cDNA (data not shown). This suggested that other peptidases might be fragments corresponding to the minimal HLA-B*5101-restricted epitope necessary to trim the N-terminal extensions to the final nonamer RU134–42 and its C-terminally extended version, RU134–47, were obtained RU134–42. We therefore sought to identify the cytosolic peptidases by annealing complementary oligonucleotides encoding the two RU1-de- involved in the N-terminal editing of the RU134–42 epitope pre- rived peptide fragments. The oligonucleotides were designed so as to re- cursors. Because we were not able to determine the relative abun- constitute a SacII site at the 5Ј end and an AvaI site at the 3Ј end, and included a stop codon immediately upstream of the AvaI site. Two addi- dance of each of the two fragments produced by the proteasome tional codons, specifying two Gly residues, were added at the 5Ј end of the due to their coelution from the HPLC columns (data not shown), minigene so as to reconstitute the exact C terminus of the Ub moiety. Upon we investigated the editing of both species. annealing, the fragments were inserted between the SacII/AvaI sites of We first focused our attention on the precursor RU1 , which pEGFP/Ub, resulting in plasmid pEGFP/Ub-RU1 and pEGFP/Ub- 29–47 34–42 carries an N-terminal extension of 5 aa, and whose C-terminal RU134–47. The same approach was used to construct the plasmid pEGFP/ ⌬G75,76 processing was studied in our previous work (Fig. 1) (16). To Ub -RU134–42 coding for an uncleavable fusion EGFP-Ub-RU134–42, except that the codons specifying the Gly at the 3Ј end of Ub were omitted. The identify the peptidase(s) involved in the N-terminal trimming of 4164 CYTOSOLIC N-TERMINAL EDITING OF ANTIGENIC PEPTIDE PRECURSORS

FIGURE 1. Sequence of the RU1 peptides used in this study. The num- bers above the sequence refer to the relevant positions within the full- length protein. The antigenic peptide is underlined, and the ﬁlled box corresponds to the constant sequence 34–47 shared by the different peptides. The open box corresponds to the sequence 34–42. TGST and STA are the two N-terminal extensions produced by the proteasome.

this precursor, we adopted the following strategy: proteasome-de- Downloaded from pleted cytosol isolated from the renal carcinoma cell line BB64- RCC was fractionated by ion-exchange chromatography. The pres- FIGURE 2. Isolation and characterization of TPP II. A, Proteasome- ence of a proteolytic activity was assayed by incubating an aliquot depleted cytosol from the renal carcinoma cell line BB64-RCC was separated by ion-exchange chromatography and eluted, in 1-ml fractions, using of each fraction with the peptide RU129–47 and by subsequent analysis by MS. The 5-aa extension at the C terminus of the an- a linear gradient from 300 to 650 mM NaCl (dotted line). Protein content was monitored by UV detection at 280 nm, in milliabsorbance units tigenic peptide sequence was included in the precursor so as to http://www.jimmunol.org/ (mAU). Fractions are separated by the vertical narrow line, and the frac- monitor for the possibility that a peptidase other than the protea- tions containing the relevant activity are marked by the thick horizontal some may directly generate the CTL-defined epitope. No fraction line (fractions 10–14). B, An aliquot of fraction 12 was incubated with the was found to generate the exact N terminus of the antigenic pep- precursor peptide RU129–47, resulting in the production of a fragment lack- ϳ tide. Rather, fractions 10–14, eluting at 420–460 mM NaCl, ing the 3 N-terminal aa (RU132–47). In each panel, the filled box corre- contained an activity that resulted in the trimming of the first 3 sponds to the sequence VPYGSFKHVDTRLQ, as described in Fig. 1. C, N-terminal aa (Fig. 2, A and B). No proteolytic activity on the Peptide RU129–47 was incubated with purified TPP II for 0 or 2 h and C-terminal extension of the antigenic peptide was detected in any subsequently analyzed by MS. After 2 h, a fragment lacking the 3 N- fraction (data not shown). Fractions 10–14 were pooled and sep- terminal aa was visible. The fragments detected with purified TPP II match those shown in B. Peaks of higher mass than RU1 correspond to salt by guest on September 23, 2021 arated by 2-D gel electrophoresis. After silver staining of the gel, 29–47 adducts. visible spots (218) were manually excised, and 77 of those were subjected to in-gel trypsin digestion. The tryptic peptide fragments were extracted from the gel and analyzed by MS. The m.w. values of the extracted peptides were introduced into the program Pro- the N-terminal processing occurred entirely within membranes, Found (http://129.85.19.192/profound_bin/WebProFound.exe) and which were absent from this preparation. used for peptide mass fingerprinting. Based on the pattern of its tryptic fragments and its migrating properties in the 2-D gel, one Puromycin-sensitive aminopeptidase generates the exact spot was unambiguously identified as TPP II. No other peptidases N terminus of RU134Ð42 were identified among the analyzed spots. TPP II is a very large To address the first hypothesis, we investigated the possibility that homomultimeric peptidase (molecular mass 5000–9000 kDa), a second cytosolic peptidase might be responsible for the final with subunit molecular mass 138 kDa, that removes tripeptides trimming of the N-terminal extension. To exclude the possibility of from the N terminus of peptides and also displays endoproteolytic contamination from peptidases located in subcellular compart- activities (30, 31). Based on indirect evidence, TPP II has been ments, proteasome-depleted cytosol isolated from erythrocytes suggested to participate in the MHC class I Ag-processing path- (these cells do not contain internal membranes (33)) was subjected way, as it may partially compensate for the generation of MHC to fractionation. The proteolytic activities of the fractions obtained class I ligands, in situations in which proteasomes are pharmaco- after separation on Q-Sepharose were tested using the fluorogenic logically inactivated (32). To confirm that the proteolytic activity peptide AAF-AMC (Fig. 3A). Two major peaks of activity were producing the fragment RU132–47 could be ascribed to TPP II, we detected in fractions 29–33 and 38–45. The activity of the first purified TPP II by immunoadsorption and assayed its activities on peak was able to digest the N terminus of RU132–47, resulting in the precursor RU129–47 (Fig. 2C). After numerous unsuccessful the enrichment of a fragment corresponding to RU134–47, which attempts to purify active rTPP II from prokaryotic as well as eu- displays the exact N terminus of the antigenic peptide (Fig. 3A, karyotic expression systems, we developed a new purification inset). In light of this result, fraction 30, the proteolytically most scheme that yielded active TPP II from 5 ϫ 107 cells. As demon- active fraction, was precipitated by TCA, separated on SDS- strated by the MS analysis, the peak profile of the digested peptide, PAGE, and stained by Coomassie blue. Each of the 12 visible using immunoadsorbed TPP II, was identical with the one detected protein bands was excised from the gel, digested by trypsin, and after incubation of the same precursor with cytosolic fraction 12 further processed as above. The tryptic peptides obtained from a (Fig. 2, compare B and C). Because the final N terminus of the protein band with apparent molecular mass 100 kDa led to the antigenic peptide was not detected, two possibilities were envis- identification of the peptidase puromycin-sensitive aminopeptidase aged: either the generation of the final N terminus was sequential (PSA). This peptidase, originally found in brain tissues (see Ref. and required another peptidase for the removal of the last 2 aa, or 34 and references therein), has recently been identified as playing The Journal of Immunology 4165

FIGURE 3. Isolation and characterization of PSA. A, The proteolytic activity of the fractions obtained after ion-exchange chromatography was tested using 100 ␮M AAF-AMC and monitoring the increased fluorescence, in arbitrary units (A.U.), emitted by the released fluorogenic group AMC (excitation/emission of 380/440 nm). Fluorescence released after incubation of AAF-AMC with unfractionated cytosol (T) or in buffer (Ϫ) is indicated on the right. Inset, Depicts the proteolytic activity of fraction 31, using RU132–47 as precursor. This activity leads to the production of peptide RU134–47. The filled box is as before. B, Peptides RU132–47 and RU129–47 were incubated with purified PSA-VSV for 0 or 2 h and subsequently analyzed by MS. After

2h,RU132–47 was completely converted into a fragment lacking the 2 N-terminal aa (middle panel). Downloaded from This fragment matches the one shown in the inset of A and corresponds to a fragment displaying the exact N terminus of the antigenic peptide. In contrast,

RU129–47 was barely degraded (right panel). The peak labeled Ϫ1 corresponds to a fragment lacking 1 N-terminal aa. See Materials and Methods for details. http://www.jimmunol.org/

a role in the N-terminal trimming of another antigenic peptide precursor that already displayed the exact C terminus of the anti- precursor (13). To ascertain the role of PSA in the final N-terminal genic peptide. Using the same assay as described above, we incu- by guest on September 23, 2021 trimming of RU134–42, we transfected and immunoadsorbed VSV- bated the precursor peptide either with immunoadsorbed TPP II tagged PSA in the human embryonic kidney cell line HEK293. (Fig. 4A) or PSA (Fig. 4B). Analysis of the digested products by Using the same technique developed for the purification of active MS revealed that both peptidases were capable of trimming the TPP II, immunoadsorbed and proteolytically active PSA was used N-terminal extension of the precursor peptide to a fragment cor- to digest the precursor peptide RU132–47. Analysis of the digested responding to the exact antigenic peptide. The dichotomy between material by MS revealed that, after 2-h incubation at 37°C, the the processing of RU129–47 and RU131–42 led us to conclude that peak corresponding to the original peptide RU132–47 had com- the sequential trimming of the first one by TPP II and PSA may be pletely disappeared and a single peak corresponding to peptide caused by the presence of particular amino acids that resist cleav-

RU134–47 could be detected (Fig. 3B). This fragment corresponds age by PSA (see Discussion). to a species displaying the exact N terminus of the antigenic peptide The N-terminal trimming of RU134Ð42 precursors is sensitive to (Fig. 1). In contrast, the precursor peptide RU129–47 was barely degraded by purified PSA during the same time frame (Fig. 3B). specific TPP II and PSA inhibitors The fractions obtained after separation on Q-Sepharose were To determine the contribution of the two identified cytosolic pep- also incubated with RU129–47, and the proteolytic activity detected tidases to the N-terminal trimming of RU134–42 precursors in un- in fractions 38–45 was compatible with the one of TPP II. How- fractionated cytosol, we performed a series of digestions of the ever, we did not manage to unambiguously identify the peptidase responsible for this activity. We concluded from these in vitro experiments that PSA trims the N-terminal extension of RU132–47 to yield the exact N terminus of the antigenic peptide. Although PSA is a peptidase with broad specificity (34), it is noteworthy that no fragment shorter than RU134–47 could be detected, indicating that PSA cannot completely degrade the peptide precursor, thereby leading to an enrichment of a peptide with the exact N terminus.

TPP II and PSA can both edit the N terminus of peptide

RU131Ð42 to its final size FIGURE 4. Both TPP II and PSA can trim RU1 . A, Purified human Because neither TPP II nor PSA alone could efficiently trim 31–42 TPP II was incubated with peptide RU131–42 for2hat37°C, and the digested RU129–47 to the exact N terminus of the CTL-defined epitope, we material was analyzed by MS. The open box corresponds to the sequence tested whether this was also the case for the second N-terminally VPYGSFKHV, displaying the final N and C terminus of the antigenic peptide. extended precursor produced by the proteasome, RU131–42 (Fig. B, Same as A, except that the precursor peptide was digested with purified 1). Contrary to the first RU1 precursor, we used in this study a PSA-VSV. 4166 CYTOSOLIC N-TERMINAL EDITING OF ANTIGENIC PEPTIDE PRECURSORS various peptide precursors in the presence or absence of specific exact N terminus of the antigenic peptide was efficiently produced

TPP II and PSA inhibitors (Fig. 5). The peptides RU129–47, in untreated cytosol (Fig. 5C) and was insensitive to butabindide RU131–42, and RU132–47 (Fig. 1) were incubated with proteasome- and only partially sensitive to AAF-CMK (F and I, respectively). depleted cytosol for 10 min at 37°C (Fig. 5, AÐC). The reaction However, no digestion was observed when the cytosol was pre- was stopped by the addition of 2% TFA and analyzed by MS. The treated with puromycin (Fig. 5L), confirming the essential role of same digestions were also performed in the presence of butabin- PSA in the final trimming of this intermediate. Finally, we ana- dide, a specific TPP II inhibitor (Fig. 5, DÐF) (35); AAF-CMK, an lyzed the trimming of the precursor RU131–42. As already pre- inhibitor of TPP II, PSA, and bleomycin hydrolase (GÐI) (13, 30); dicted from the result shown in Fig. 4, a fragment corresponding to and puromycin, a specific PSA inhibitor (JÐL) (34). In the case of the final size of the antigenic peptide was produced both in un-

RU129–47, a fragment displaying the final N terminus of the anti- treated cytosol and in cytosol treated with inhibitors (Fig. 5, B, E, genic peptide was readily detectable after incubation with un- H, and K). We conclude that this precursor can be edited simul- treated cytosol (Fig. 5A). No such fragment could be detected taneously either by TPP II or PSA, but the contribution of yet when the peptide was incubated with cytosol pretreated with but- another peptidase on the editing of this particular peptide cannot be abindide or AAF-CMK (Fig. 5, D and G, respectively). Interest- ruled out, as peaks corresponding to fragments lacking 1 and 2 ingly, not only was the fragment with the exact N terminus absent, N-terminal aa could be detected in all conditions. but the intermediate corresponding to RU132–47 was not detectable either. The result obtained after treatment with butabindide con- firms and extends the findings shown in Figs. 2 and 3, namely that The N-terminal processing of RU134Ð42 precursors occurs

predominantly in the cytosol Downloaded from the processing of the N-terminal extension of RU129–47 in unfractionated cytosol is a sequential process that requires the activity of Several reports have suggested that the N-terminal trimming of anti- TPP II. Finally, puromycin-treated cytosol led to an accumulation genic peptides may occur within the ER. Although we presented of the intermediate RU132–47 by blocking the second proteolytic evidence that the processing of RU134–42 precursors could occur in event that normally leads to the generation of the ﬁnal N terminus the cytosol (Fig. 5), we nevertheless tested whether the trimming (Fig. 5J). could also take place within ER membranes. We puriﬁed mem-

Peptide RU132–47, which corresponds to the fragment produced branes from the BB64-RCC cell line, separated integral membrane http://www.jimmunol.org/ after the digestion of the longer precursor by TPP II, was also proteins from soluble luminal proteins by Triton X-114 extraction incubated with cytosol. As expected, the fragment displaying the (see Materials and Methods for details), and tested the proteolytic by guest on September 23, 2021

FIGURE 5. Effects of TPP II and PSA inhibitors on the processing of RU1 precursor peptides. Pep- tides RU129–47 (A, D, G, and J), RU131–42 (B, E, H, and K), and RU132–47 (C, F, I, and L) were incubated with unfractionated proteasome-depleted cytosol of BB64-RCC for 10 min at 37°C. Reactions were stopped by adding 2% TFA. The samples were lyophilized and analyzed by MS. For peptide

RU129–47 (A, D, G, and J), only the region of the spectrum encompassing RU132–47 and RU134–47 is shown. AÐC, Peptides digested by untreated cytosol. DÐF,AsAÐC, but in presence of 5 ␮M butabindide, a speciﬁc inhibitor of TPP II. GÐI,AsAÐC, but in presence of 50 ␮M AAF-CMK, a second inhibitor of TPP II. JÐL,AsAÐC, but in presence of 50 ␮M puromycin, a speciﬁc inhibitor of PSA. The stars identify contaminating peaks (already present at time 0, not shown). In B, E, H, and K, the peaks labeled Ϫ1 and Ϫ2 correspond to fragments lacking 1 and 2 N-terminal aa. See text for details. The Journal of Immunology 4167 Downloaded from

FIGURE 6. The N-terminal trimming of RU134–42 precursors occurs mainly in cytosolic extract. BB64-RCC cells were separated into cytosol and membranes. The microsomal fraction was further fractionated so as to separate membrane-associated proteins (fraction B) from soluble luminal proteins http://www.jimmunol.org/

(fraction A). An aliquot of each fraction was used to digest RU129–47 (A, D, and G), RU132–47 (B, E, and H), and RU131–42 (C, F, and I). AÐC, Fragments obtained after digestion of the precursors with incomplete cytosol. As in Fig. 6, the stars identify contaminating peaks. DÐF, Same as AÐC with fraction A, containing soluble luminal proteins. GÐI, Same as AÐC with fraction B, containing membrane-associated proteins. The peaks ranging from mass 800 to 1000 (I) correspond to the mass of detergent micelles. The filled and open boxes are as before. J, Proteolytic activity of the various fractions was tested using 100 ␮M AAF-AMC and monitoring the increased fluorescence, in arbitrary units (A.U.), emitted by the released fluorogenic group. Where indicated, the fractions were preincubated with 5 ␮M butabindide and 50 ␮M puromycin before addition of the fluorogenic substrate. K, Western blot analysis of the various fractions detecting the presence of TPP II and PSA. See text for details. activity of each of these fractions using the three peptide precur- one other peptidase can digest this peptide. Indeed, as already de- by guest on September 23, 2021 sors. As before, we could clearly detect proteolytic cleavage of all tected in Fig. 5, B, E, H, and K, peaks corresponding to fragments precursors in the cytosolic fraction, resulting in the formation of a missing 1or 2 N-terminal aa could also be observed in digested fragment displaying the exact N terminus of the antigenic peptide material obtained from fraction A (Fig. 6F). This proteolytic ac- (Fig. 6, AÐC). Very little proteolytic activity was detected in the tivity is insensitive to butabindide, puromycin, and AAF-CMK fraction A, containing soluble luminal proteins (Fig. 6, DÐF), and (Fig. 5). It is noteworthy that both other precursors do not seem to no activity was detected in the fraction B, containing membrane- be substrate of this peptidase. We do not have any information on embedded proteins (Fig. 6, GÐI). Proteolytic activity present in the this peptidase at the moment. three fractions was also independently monitored by the release of AMC from the fluorogenic tripeptide AAF-AMC (Fig. 6J). The The presentation of RU134Ð42/HLA-B51 is blocked by cytosolic extract was proteolytically active against AAF-AMC, lactacystin and AAF-CMK and these activities could be partially blocked either by butabin- In an attempt to correlate our results obtained in an acellular sys- dide or puromycin, indicating that: 1) TPP II and PSA are present tem with the cellular pathway leading to the presentation of in this fraction, and 2) other peptidases are also active against this RU134–42 by HLA-B51, we treated BB64-RCC with specificin- fluorogenic peptide. The presence of the two peptidases was fur- hibitors and tested their recognition by CTL. Cells treated with the ther confirmed by Western blot analysis (Fig. 6K). Some proteo- proteasome inhibitor lactacystin were poorly recognized by spe- lytic activity could also be detected in fraction A. However, this cific CTL, as indicated by the low level of TNF produced by the activity was totally resistant to puromycin (confirming the absence CTL clone (Fig. 7). Recognition was partially restored when the of PSA from this fraction), but was completely blocked by but- antigenic peptide was added exogenously. In comparison, un- abindide, indicating the presence of contaminating TPP II in this treated cells were efficiently recognized by the same CTL clone, fraction. This was independently confirmed by Western blot anal- confirming that the proteasome played an essential role in the pre- ysis using an anti-TPP II Ab (Fig. 6K). The presence of TPP II sentation of this CTL-defined epitope. We also tested whether the could be responsible for the small peak of RU132–47 and RU132–42 recognition of RU134–42/HLA-B51 was influenced by the pres- detected in D and F, respectively. The presence of TPP II in frac- ence of the inhibitor AAF-CMK. As with lactacystin, cells treated tion A could be explained by the fact that a fraction of this very with AAF-CMK were poorly recognized by specific CTL. Again, large peptidase (5–9 Md) cosediments with microsomal mem- exogenously added peptide led to a partial restoration of the rec- branes. Indeed, we found that a 60-min centrifugation at ognition of BB64-RCC cells. The lack of recognition resulting 350,000 ϫ g efficiently sediments most of the TPP II present in the from the AAF-CMK treatment could not be ascribed to an inhibi- cell lysate (data not shown). tion of proteasome because, as tested by us and reported by others,

A close analysis of the fragments produced from RU131–42 by proteasomal activities were not inﬂuenced by AAF-CMK (13, 30) the proteolytic activity present in fraction A suggests that at least (data not shown). In our hands, 100 ␮M lactacystin did not inhibit 4168 CYTOSOLIC N-TERMINAL EDITING OF ANTIGENIC PEPTIDE PRECURSORS

FIGURE 7. Lactacystin and AAF-CMK block the recognition of tumor cells by speciﬁc CTL. BB64-RCC cells were treated with the proteasome inhibitor lactacystin and the protease inhibitor AAF-CMK or left untreated. CTL clone 381/84 was added to the cells, and TNF release was measured (open bars). As control, saturating amounts of the antigenic peptide

RU134–42 were added exogenously (filled bars). Downloaded from the activity of TPP II (data not shown). Taken together, these results indicate that, in cells, the proteasome most likely does not directly produce the final antigenic peptide RU134–42, but that additional peptidases, like TPP II and/or PSA, are necessary to gen- FIGURE 8. Peptide RU134–42 is transported by the TAP complex. A, erate the antigenic peptide. We were unable to test the effect of HEK293-EBNA cells were transfected with a plasmid coding for HLA- http://www.jimmunol.org/ butabindide on the presentation of RU134–42 because this drug B51 and constructs containing the RU1 full-length cDNA or minigenes does not cross the cell membrane (data not shown). Puromycin coding for the CTL-defined epitope RU134–42 with a C-terminal extension could not be tested either, due to its excessive cell toxicity (data (pEGFP/Ub-RU134–47) or the minimal epitope RU134–42 (pEGFP/Ub- not shown). RU134–42). The peptides encoded by the latter two constructs are produced as a result of the proteolytic cleavage after the last residue of Ub, a system Cells transfected with a minigene encoding the minimal that allows to bypass the need for an N-terminal methionine. Some of the cells were also transfected with a plasmid encoding the herpes simplex antigenic peptide RU134Ð42/HLA-B51 are efficiently recognized by specific CTL protein ICP47, which blocks the TAP-mediated transport of antigenic peptides. The presentation of RU1 by HLA-B51 in absence (open bars) or

34–42 by guest on September 23, 2021 The antigenic peptide RU134–42 contains a subaminoterminal P presence (filled bars) of ICP47 was monitored by measuring the amount of (Fig. 1) and is thus predicted to be poorly transported by TAP in TNF-␣ released by CTL 381/84 incubated for 24 h with the transfected its final form. However, we have shown that the production of the cells. B, HEK293 cells expressing EGFP-Ub-RU134–47 and EGFP- ⌬G75,76 35 final N terminus can occur in the cytosol. To test whether the Ub -RU134–47, respectively, were metabolically labeled with S and N-terminally trimmed antigenic peptide can be transported into lysed, and the lysate was immunoprecipitated, using an Ab against a pep- the ER, the human embryonic kidney cells HEK293 were tran- tide sequence located between the C terminus of EGFP and the N terminus siently transfected with the cDNA-encoding HLA-B51 and either of Ub. The precipitated material was separated by SDS-12% PAGE and subjected to autoradiography. As control, EGFP-Ub was immunoprecipi- the RU1 cDNA or two minigenes coding for, respectively, peptide tated from radiolabeled cells expressing the 118-aa-long protein Melan-A RU134–47, displaying the exact N terminus, but carrying a C-ter- instead of the RU1 peptides. The arrow indicates the position of EGFP-Ub, minal extension, and RU134–42, the minimal HLA-B51-restricted and the star indicates unidentified species. See text for details. epitope (Fig. 1). The vectors used for the expression of the minigenes were designed based on the ubiquitin/protein/reference technique described previously (22, 36). In short, the plasmid codes for anymore, confirming the necessity of functional TAP for the rec- the tripartite linear fusion protein EGFP-ubiquitin (Ub) minigene. ognition of target cells (Fig. 8A). The slightly reduced inhibition of

The EGFP-Ub moiety is cotranslationally cleaved after the last the recognition of cells expressing the minimal epitope (RU134–42) residue of Ub by the cytosolic Ub peptidase, thereby liberating the in presence of ICP47 could be explained by the incomplete effects peptides in the cytosol (37, 38). In addition, this gene arrangement of ICP47 on TAP, a TAP-independent peptide transport, or the fact also allows the expression of minigenes without the need of the that a small percentage of the peptide may be released into the initiation codon for methionine. As shown in Fig. 8A, cells trans- extracellular medium and recaptured by HLA-B51 molecules at fected with the three RU1 constructs were recognized by the spe- the cell surface. In other experiments, the effect resulting from the cific CTL. It is noteworthy that the cells transfected with the vector expression of ICP47 was confirmed to be specific, and not due to encoding the minimal antigenic peptide sequence were more effi- dilution by the additional plasmid DNA transfected (data not ciently recognized by the CTL, supporting the notion that the pro- shown). We conclude from these experiments that the antigenic teasomal processing may limit the efficiency of Ag presentation. peptide RU134–42, carrying P at position 2, can be translocated More efficient recognition of DNA-encoded RU134–42 occurred across the ER membrane in its final form via the TAP transporters. when cells were transfected with plasmid concentrations ranging We could exclude that the fusion GFP-Ub could be cleaved within from 1.25 to 50 ng (data not shown). To confirm that the antigenic the ER because it lacks an ER-targeting signal sequence and be- peptide was transported by the TAP complex in our experimental cause the Ub-specific peptidase is absent from the ER (38) (F. conditions, the gene encoding the natural TAP inhibitor ICP47 Le´vy, unpublished data). from herpes simplex was cotransfected with the plasmids de- We also tested the possibility that a significant amount of un- scribed above (39). As expected, these cells were not recognized cleaved EGFP-Ub peptide could be present in transfected cells and The Journal of Immunology 4169 could lead to the production of N-terminally extended antigenic face expression of MHC class I and HPLC profile of peptides peptides. Therefore, we compared, by SDS-PAGE, the mobility of eluted from MHC class I molecules, suggested that TPP II may two EGFP-Ub minigene constructs. The first one codes for EGFP- compensate for the lack of proteasome activity (32, 41). However,

Ub-RU134–47, which is cleaved by Ub-specific peptidase to pro- the exact contribution of TPP II to this process remains to be duce EGFP-Ub and RU134–47. The second plasmid encodes elucidated, as the activities of the proteasome may not be com- ⌬G75,76 EGFP-Ub -RU134–42, which lacks the two C-terminal Gly pletely blocked in those cells (42). Another study demonstrated residues of Ub, thereby producing an uncleavable form of Ub. that TPP II did not only remove tripeptides from the N terminus of Indeed, the C-terminal Gly have been shown to be essential for the peptides, but also had some endoproteolytic activities, which could Ub peptidase-mediated cleavage of Ub (40). Both constructs con- potentially produce antigenic peptides (30). Of note is that in this tained a sequence derived from the influenza A HA between the C study we did not observe any other endoproteolytic activity of terminus of EGFP and the N terminus of Ub, which can be rec- human TPP II than the one removing tripeptides from free N ter- ognized by a specific Ab (22). Cells were transfected with both mini (data not shown). However, in none of the two cases men- plasmids, metabolically labeled with [35S]Met/Cys, and lysed in tioned above was TPP II directly identified in the trimming of a the presence of alkylating agent to prevent postlysis deubiquityla- specific antigenic peptide precursor. Contrary to the role of TPP II tion, and the cleared lysate was immunoprecipitated using the anti- in the production of antigenic peptides, the contribution of PSA to HA mAb linked to protein G-Sepharose. The length of the RU1 the processing of a CTL-defined epitope was recently revealed by minigenes was selected so as to maximize the migration differ- an experimental approach similar to the one described in this work ences after separation on SDS-PAGE. As shown in Fig. 8B, the (13). In that approach, PSA was shown to degrade N-terminally Downloaded from SDS mobility of EGFP-Ub-RU134–47 carrying the wild-type Ub extended precursors of the antigenic peptide VSV nuclear pro- moiety was indistinguishable from EGFP-Ub derived from a plas- tein52–59 and to generate, among many other fragments, a peptide mid encoding a melanoma-associated protein fused to EGFP-Ub. with the final N terminus. Using the precursor peptide RU129–47, ⌬G75,76 In contrast, EGFP-Ub -RU134–42 migrated with a slightly we showed that the production of the final N terminus of the an- slower mobility. This result indicated that the cleavage at the Ub tigenic peptide was an ordered process, which required the sequen- minigene junction had occurred in the wild-type construct and pro- tial activities of the peptidases TPP II and PSA (Figs. 3, 4, and 6). duced a polypeptide that differs from the mutant construct by 7 aa. This was also the case in a cytosolic extract, which contained other http://www.jimmunol.org/ A very faint band with slower mobility could be observed for the active peptidases (Figs. 6 and 7J). A molecular explanation for this nonmutated construct, but its precise identity could not be deter- resides probably in the specificity of the individual peptidases. It mined because a band of identical mobility was also visible in the appears that neither TPP II nor PSA can cleave a peptide bond construct carrying the mutated Ub and could be sometimes de- before or after Pro (P). In addition, PSA does not cleave efficiently tected in the EGFP-Ub control plasmid. Even though the vast ma- peptide bonds involving a Gly (G) residue (34). Because the se- jority of EGFP-Ub-RU134–42 molecules was cleaved, the possi- quence of the precursor peptide is TGSTAVP. . . , in which VP bility that a small fraction of EGFP-Ub-RU134–42 may not be corresponds to the first 2 aa of the CTL-defined epitope (Fig. 1), cleaved by Ub-specific proteases and produces an amino-termi- this may explain why TPP II has to remove the first 3 aa (it cannot nally extended antigenic peptide precursor cannot be completely cleave further because this would involve the residue P) before by guest on September 23, 2021 ruled out. Nevertheless, the data presented in this work support the PSA, which does not cleave the full-length precursor because of notion that the final processing of RU134–42 can take place in the the amino acid G, can trim the last 2 aa. PSA will then stop at the cytosol. exact N terminus of the antigenic peptide because of the residue P. Such a process may be specific for antigenic peptides carrying the Discussion residue P at position 2, as is the case for certain ligands of the In this work, we investigated the N-terminal trimming of the two HLA-B7, HLA-B8, HLA-B15, HLA-B51, and other class I mol-

RU134–42 precursor peptides, RU129–42 and RU131–42, liberated ecules. However, the fact that the same peptidase (PSA) has been by the proteasome in vitro. We show that two cytosolic peptidases identiﬁed using two different peptide precursors may not be coin- can trim the N-terminal extensions of these peptide precursors. The cidental, and leads us to postulate that a limited number of pepti- peptidases, TPP II and PSA, acted sequentially on peptide dases will be responsible for the ﬁnal editing of MHC class I

RU129–42 and redundantly on peptide RU131–42. In all cases, this ligands. At present, we do not know whether this sequence of process led to the transient accumulation of a species displaying action is identical in all cell types nor in situations in which the the final N terminus of the antigenic peptide. Moreover, we expression of other peptidases is induced, as is the case for leucyl showed that proteasome and TPP II/PSA inhibitors blocked the aminopeptidase induced by IFN-␥ (15). ϩ presentation of RU134–42 by HLA-B51 tumor cells. Taken to- The production of the final antigenic peptide from the N-termi- gether, these data suggest that the production of the epitope nally extended precursor RU131–42 could be achieved by TPP II RU134–42/HLA-B51 may be a cytosolic process involving at least and PSA in a redundant fashion. This result is in agreement with three distinct peptidases, the proteasome, TPP II, and PSA. the findings reported on the generation of the N-terminal end of

The N-terminal trimming peptidases identified to date share the VSV nuclear protein52–59, namely that the final N terminus of this properties of having broad specificities, of being redundant, and of antigenic peptide could be obtained by two redundant processes, trimming antigenic peptide precursors without detectable accumu- mediated either by PSA or, in that case, by bleomycin hydrolase lation of a species with the correct N terminus. We were therefore (13). The likely explanation for the different processing of surprised that the generation and the transient accumulation of the RU131–42 and RU129–47 resides in the length and the sequence of final N terminus of RU134–42 from RU129–47 required the sequen- the N-terminal amino acid extension. Therefore, the choice of pep- tial action of two distinct peptidases. The two identified peptidases, tidase(s) responsible for the postproteasomal trimming of antigenic TPP II and PSA, have already been implicated, in different exper- peptide precursors may be dictated by the nature of the N-terminal imental conditions, in the MHC class I Ag-processing pathway. extension produced by the proteasome. We have analyzed the pro- The first one, TPP II, was identified using cells that had been cessing of several antigenic peptide precursors in vitro and have adapted to a proteasome inhibitor (32). This treatment induced the noticed that the proteasome can, in some instances, directly gen- overexpression of TPP II, and indirect evidences, such as cell sur- erate the exact antigenic peptide (our unpublished data). However, 4170 CYTOSOLIC N-TERMINAL EDITING OF ANTIGENIC PEPTIDE PRECURSORS this species is frequently accompanied by the presence of peptides 4. Lucchiari-Hartz, M., P. M. van Endert, G. Lauvau, R. Maier, A. Meyerhans, that carry additional N-terminal amino acids. It will be interesting D. Mann, K. Eichmann, and G. Niedermann. 2000. Cytotoxic T lymphocyte epitopes of HIV-1 Nef: generation of multiple definitive major histocompatibility to study whether the final antigenic peptide produced within a cell complex class I ligands by proteasomes. J. Exp. Med. 191:239. will be more efficiently loaded onto MHC class I molecules than 5. Shastri, N., S. Schwab, and T. Serwold. 2002. Producing nature’s gene-chips: the generation of peptides for display by MHC class I molecules. Annu. Rev. Immu- the precursors requiring further N-terminal processing. nol. 20:463. N-terminal trimming in the ER has also been described (5). 6. Komlosh, A., F. Momburg, T. Weinschenk, N. Emmerich, H. Schild, E. Nadav, However, because no specific peptidase has been identified at the I. Shaked, and Y. Reiss. 2001. A role for a novel luminal endoplasmic reticulum aminopeptidase in final trimming of 26 S proteasome-generated major histocom- molecular level, it is difficult to speculate on the role of this ER patibility complex class I antigenic peptides. J. Biol. Chem. 276:30050. trimming. The fact that most peptides isolated from the HLA-A2 7. Serwold, T., S. Gaw, and N. Shastri. 2001. ER aminopeptidases generate a unique molecules of the TAP-deficient cell line T2 are considerably pool of peptides for MHC class I molecules. Nat. Immun. 2:644. 8. Lobigs, M., G. Chelvanayagam, and A. Mu¨llbacher. 2000. Proteolytic processing longer in size than those isolated from normal cells (43) indicates of peptides in the lumen of the endoplasmic reticulum for antigen presentation by that if aminopeptidases are active in the ER, they may be very major histocompatibility class I. Eur. J. Immunol. 30:1496. specific or play a minor role in the processing of antigenic peptides 9. Paz, P., N. Brouwenstijn, R. Perry, and N. Shastri. 1999. Discrete proteolytic intermediates in the MHC class I antigen processing pathway and MHC I-de- derived from signal sequences released by the signal peptidase in pendent peptide trimming in the ER. Immunity 11:241. the ER. In this study, we directly searched for membrane-embed- 10. Snyder, H. L., I. Bacik, J. R. Bennink, G. Kearns, T. W. Behrens, T. Ba¨chi, M. Orlowski, and J. W. Yewdell. 1997. Two novel routes of transporter associ- ded or luminal peptidases that would mediate the final trimming of ated with antigen processing (TAP)-independent major histocompatibility com- our precursor (Fig. 7). Although we could detect a proteolytic ac- plex class I antigen processing. J. Exp. Med. 186:1087. tivity other than the one mediated by TPP II in our membrane 11. Craiu, A., T. Akopian, A. Goldberg, and K. L. Rock. 1997. Two distinct proteo-

lytic processes in the generation of a major histocompatibility complex class Downloaded from preparation, it was only acting on the precursor RU131–42 and was I-presented peptide. Proc. Natl. Acad. Sci. USA 94:10850. also found in the cytosol. Moreover, this activity was not inhibited 12. Hammond, S. A., R. P. Johnson, S. A. Kalams, B. D. Walker, M. Takiguchi, by AAF-CMK even though this drug efficiently blocked the CTL J. T. Safrit, R. A. Koup, and R. F. Siliciano. 1995. An epitope-selective, transporter associated with antigen presentation (TAP)-1/2-independent pathway and recognition of APCs, suggesting that this peptidase is not partic- a more general TAP-1/2-dependent antigen-processing pathway allow recognition of the HIV-1 envelope glycoprotein by CD8ϩ CTL. J. Immunol. 154:6140. ipating to the final processing of RU134–42. One reason for this is that this peptidase is localized in another subcellular compartment, 13. Stoltze, L., M. Schirle, G. Schwarz, C. Schro¨ter, M. W. Thompson, L. B. Hersh, H. Kalbacher, S. Stevanovic, H.-G. Rammensee, and H. Schild. 2000. Two new which contaminated our microsomal preparation. Alternatively, proteases in the MHC class I processing pathway. Nat. Immun. 1:413. http://www.jimmunol.org/ 14. Silva, C. L., F. C. Portaro, V. L. Bonato, A. C. de Camargo, and E. S. Ferro. 1999. the product RU131–42 detected in our in vitro digestion assay may Thimet oligopeptidase (EC 3.4.24.15), a novel protein on the route of MHC class constitute only a minor species that is not produced intracellularly. I antigen presentation. Biochem. Biophys. Res. Commun. 255:591. Irrespective of the role of this peptidase in this process, our data 15. Beninga, J., K. L. Rock, and A. L. Goldberg. 1998. Interferon-␥ can stimulate suggest that the antigenic peptide RU1 is made in the cytosol post-proteasomal trimming of the N terminus of an antigenic peptide by inducing 34–42 leucine aminopeptidase. J. Biol. Chem. 273:18734. and can be transported as such across the ER membrane. 16. Morel, S., F. Le´vy, O. Burlet-Schiltz, F. Brasseur, M. Probst-Kepper, A.-L. Pei- After prolonged incubation of the various RU134–42 precursors trequin, B. Monsarrat, R. Van Velthoven, J.-C. Cerottini, T. Boon, et al. 2000. in cytosol, the peptides were eventually completely degraded (data Processing of some antigens by the standard proteasome but not by the immu- noproteasome results in poor presentation by dendritic cells. Immunity 12:107. not shown). This may be explained by the fact that our in vitro 17. Rammensee, H.-G., J. Bachmann, N. P. N. Emmerich, O. A. Bachor, and digestion assay does not contain any membrane, which, in cells, S. Stevanovic. 1999. SYFPEITHI: database for MHC ligands and peptide motifs. by guest on September 23, 2021 can offer a physical barrier against the attack of other cytosolic Immunogenetics 50:213. 18. Gubler, B., S. Daniel, E. A. Armandola, J. Hammer, S. Caillat-Zucman, and peptidases. However, the mechanism by which antigenic peptides P. M. van Endert. 1998. Substrate selection by transporters associated with an- produced in their final form in the cytosol reach the TAP trans- tigen processing occurs during peptide binding to TAP. Mol. Immunol. 35:427. 19. Uebel, S., W. Kraas, S. Kienle, K.-H. Wiesmu¨ller, G. Jung, and R. Tampe´. 1997. porters has not been elucidated. Interestingly, a membrane-asso- Recognition principle of the TAP transporter disclosed by combinatorial peptide ciated protein of 100 kDa, p100, has been identified, which binds libraries. Proc. Natl. Acad. Sci. USA 94:8976. peptides, possibly transiently, on the cytoplasmic side of the ER 20. Van Endert, P. M., D. Riganelli, G. Greco, K. Fleischhauer, J. Sidney, A. Sette, and J. F. Bach. 1995. The peptide-binding motif for the human transporter as- membrane (44). The association between this protein and the sociated with antigen processing. J. Exp. Med. 182:1883. membrane appears to be mediated by an unknown protein. Be- 21. Lauvau, G., K. Kakimi, G. Niedermann, M. Ostankovitch, P. Yotnda, H. Firat, cause the m.w., pI, and peptide interaction properties of p100 are F. V. Chisari, and P. M. van Endert. 1999. Human transporters associated with antigen processing (TAPs) select epitope precursor peptides for processing in the similar to those of PSA, it is tempting to speculate that p100 could endoplasmic reticulum and presentation to T cells. J. Exp. Med. 190:1227. be PSA. If this were the case, it would provide a mean to simul- 22. Valmori, D., U. Gileadi, C. Servis, P. R. Dunbar, J.-C. Cerottini, P. Romero, taneously edit the N terminus of longer peptide precursors and V. Cerundolo, and F. Le´vy. 1999. Modulation of proteasomal activity required for the generation of a CTL-defined peptide derived from the tumor antigen increase the chance of having the final antigenic peptide trans- MAGE-3. J. Exp. Med. 189:895. ported by the TAP1/2 complex. Experiments aimed at elucidating 23. Tobler, A. R., D. B. Constam, A. Schmitt-Gra¨ff, U. Malipiero, R. Schlapbach, and A. Fontana. 1997. Cloning of the human puromycin-sensitive aminopeptidase the intracellular localization of PSA are underway. and evidence for expression in neurons. J. Neurochem. 68:889. 24. Tomkinson, B., and F. Nyberg. 1995. Distribution of tripeptidyl-peptidase II in the central nervous system of rat. Neurochem. Res. 20:1443. Acknowledgments 25. Shevchenko, A., M. Wilm, O. Vorm, and M. Mann. 1996. Mass spectrometric We thank Dr. A. Fontana for the generous gift of the plasmid coding for sequencing of protein silver-stained polyacrylamide gels. Anal. Chem. 68:850. human PSA-VSV, Dr. H.-G. Rammensee for the plasmid encoding ICP47, 26. Gharahdaghi, F., C. R. Weinberg, D. A. Meagher, B. S. Imai, and S. M. Mische. Dr. J.-C. Schwartz for butabindide, Dr. J.-C. Cerottini and L. Chapatte for 1999. 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