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* Ludwig Institute for Cancer Research, Lausanne Branch, and
Institute of Biochemistry, University of Lausanne, Epalinges, Switzerland;
Ludwig Institute for Cancer Research, Brussels Branch, Université Catholique de Louvain, Brussels, Belgium;
DIBIT, San Raffaele Scientific Institute, Milan, Italy; and
¶ Ludwig Institute for Cancer Research, Uppsala Branch, Biomedical Center, Uppsala, Sweden
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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An essential step in the production of the MHC class I-restricted peptides is the degradation of proteins by the proteasome (1). The proteasome is a large multicatalytic protease, present in the cytosol and the nucleus of eukaryotic cells, which degrades the bulk of intracellular proteins and generates peptides ranging from 3 to 22 aa in length (2). Whereas some antigenic peptides are directly produced by the proteasome in their final form, others are produced as precursor peptides (3, 4). Those precursor peptides display the exact C terminus of the final antigenic peptides, but carry N-terminal amino acid extensions of various lengths. It is therefore assumed that other peptidases, in the endoplasmic reticulum (ER)5 or in the cytosol, are involved in the N-terminal trimming of precursor peptides (5). Although ER resident proteases have been inferred to function in the Ag-processing pathway, none have yet been described at the molecular level (6, 7, 8, 9, 10, 11, 12). In contrast, several cytosolic peptidases potentially involved in this process have been identified. Those include puromycin-sensitive aminopeptidase, bleomycin hydrolase, thimet oligopeptidase, and leucyl aminopeptidase (13, 14, 15). A common feature of these peptidases is that they display a broad specificity and do not lead to an enrichment of the exact antigenic peptide. In some instances, the generation of the accurate N terminus of antigenic peptides can be mediated by more than one peptidase in a redundant manner (13).
The human gene RU1 codes for a ubiquitously expressed intracellular protein of unknown function. A CTL clone, originally raised against an autologous renal carcinoma, recognized an HLA-B51-restricted peptide derived from the RU1 region spanning aa 34–42 (RU134–42), with sequence VPYGSFKHV (16). In accordance 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 terminus of this CTL-defined epitope was directly produced by the standard proteasome, but not the final N terminus. Consequently, other peptidases could act on the N-terminal extension to produce the antigenic nonamer. Based on several in vitro studies indicating that antigenic peptides containing a subaminoterminal P are poor substrates for the human TAP transporters (7, 18, 19, 20), it has been inferred that such peptides are transported through the TAP as N-terminal extended precursors and that the final N terminus is generated by peptidases localized within the ER (7, 21).
Contrary to this prediction, our results indicate that the antigenic peptide RU134–42, which contains P at second position, can be produced in the cytosol, before TAP-mediated transport. By using a substrate-based assay, we identified two cytosolic peptidases that trim the N terminus of the RU134–42 precursors produced by the proteasome. These two peptidases, tripeptidyl peptidase II (TPP II) and puromycin-sensitive aminopeptidase (PSA), act on the N-terminally extended precursors to produce, and transiently enrich for, the exact N terminus of the antigenic peptide. We also searched for proteolytic activities in membranes enriched in ER. Although we observed a detectable proteolytic activity against one of the proteasomal products, RU131–42, it could apparently not rescue the loss of CTL recognition of tumor cells resulting from the inhibition of proteasome and TPP II/PSA activities. Our data suggest that the production of RU134–42/HLA-B51 is a cytosolic process, involving the proteasome to generate the exact C terminus of the antigenic peptide and TPP II and PSA to trim the N-terminal extensions produced by the proteasome.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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For the identification of TPP II, BB64-renal cell carcinoma (RCC) (6.3 x 108 cells) were mechanically disrupted by douncing in a Dounce homogenator. Sucrose was immediately added to the homogenate to a final concentration of 250 mM. Debris were removed by centrifugation at 13,000 x g for 15 min at 4°C. The supernatant was transferred to ultracentrifuge tubes and subjected to ultracentrifugation at 80,000 x g for 45 min at 4°C. Proteasomes were removed from the clear supernatant (complete cytosol) by affinity purification, using the mAb anti-proteasome MCP21 (22). Proteasome-depleted cytosol was loaded onto a high performance ion-exchange 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 Tris-HCl, pH 7.6), washed with five-column volumes of buffer A, and pre-eluted with 30% buffer B (20 mM Tris-HCl, pH 7.6, 1 M NaCl). The adsorbed material was then eluted, using a linear gradient of 30–70% buffer B, in 35 fractions of 1 ml each. An aliquot of each fraction (50 µl) was incubated with 4 nmol peptide RU129–47 (TGSTAVPYGSFKHVDTRLQ, in one-letter code, in which the underlined sequence corresponds to the final CTL-defined antigenic peptide) for 1 h at 37°C. The reaction was stopped by adding trifluoroacetic acid (TFA) to a final concentration of 2%. The samples were then analyzed by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (MS), as previously described (22). The positive fractions (fractions 10–14) were pooled, concentrated to a final volume of 500 µl, and precipitated with 20% TCA overnight at 4°C. The precipitated proteins were pelleted at 13,000 x g for 10 min at 4°C, washed three times with 100% cold (-20°C) acetone, and lyophilized. The proteins were then subjected to two-dimensional gel electrophoresis (2-D gel).
For the identification of PSA, proteasome-depleted cytosol from 100 ml concentrated human erythrocytes was loaded onto a DEAE-32 column, pre-equilibrated in 10 mM phosphate buffer, pH 7.5. The column was extensively washed, and the adsorbed material was eluted in 30 x 1-ml fractions with 200 mM phosphate buffer, pH 7.5. The eluted material was then desalted on a Sephacryl S-300 column, in 20 mM Tris-HCl, pH 7.6, and loaded onto a Source 15Q PE 4.6/100 column, at a flow of 1 ml/min. After extensive washing with buffer A, the adsorbed material was eluted in 60 x 1-ml fractions, using a linear gradient of 0–65% buffer B. An aliquot (20 µl) of each fraction was tested with 100 µM H-Ala-Ala-Phe-amidomethylcoumarin (AAF-AMC) for 15 min at 37°C. Proteolytic activity was monitored by the increased fluorescence resulting from the release of the AMC group (excitation/emission 380/440 nm). A total of 4 nmol peptide RU132–47 was incubated with 12 µl fractions 29, 31, and 32, respectively, for 20 min at 37°C. The digestion was stopped with 2% TFA and analyzed by MS as above. Fraction 30 was precipitated with 20% TCA and processed as above. After lyophilization, the protein pellet was resuspended in SDS-sample buffer containing DTT and boiled for 3 min at 95°C. After cooling, iodoacetamide was added to the samples before separation by SDS-12% PAGE. The gel was stained with 0.2% Coomassie brilliant blue R in 20% methanol/0.5% acetic acid and destained with 20% methanol, and the visible bands were excised and treated, as described below.
TPP II was purified from 5 x 107 HEK293 cells by affinity purification, using 5 µl polyclonal chicken Ab anti-human TPP II (Immunsystem, Uppsala, Sweden), 5 µg anti-chicken biotin conjugate Ab (Promega, Madison, WI), and 20 µl streptavidin-coated agarose beads (Pierce, Rockford, IL). The plasmid PSA-vesicular stomatitis virus (VSV), directing the synthesis of PSA carrying a C-terminal Ab epitope from the vesicular stomatitis virus (a generous gift of A. Fontana, University Hospital, Zürich, Switzerland), was transiently transfected into 4 x 107 HEK293 cells using Fugene (Roche, Basel, Switzerland) and the manufacturer-supplied protocol. Twenty-four hours posttransfection, cells were lysed in 1% Triton X-100, and PSA-VSV was immunoprecipitated using 5 µg anti-VSV tag mAb (Fluka, Buchs, Switzerland) and 20 µl protein G-Sepharose slurry (Pierce). Due to the limited number of cells used in this assay, the final purity of the isolated peptidases could not be ascertained. However, the specificities of both Abs have been described by others (23, 24), and the proteolytic activities of the TPP II and PSA preparations could be completely blocked by butabindide and puromycin, respectively (data not shown). The immunoprecipitated material was washed four times and was used to digest 4 nmol peptides of interest. At the end of the digestion, the supernatant was collected and the digestion products were analyzed by MS.
Preparative 2-D gel electrophoresis
The protein pellets obtained from the above-mentioned fractions
10–14 were resuspended in 600 µl loading buffer (40 mM Tris-HCl, pH
8.0, 8 M urea, 4% CHAPS
(3-[(3-cholamidopropyl)dimethylammonio]-1-propane-sulfonate), 65 mM
dithioerythritol, 0.01% bromphenol blue), loaded onto 18-cm-long
nonlinear, pH 3–10 gradient strips (Amersham Pharmacia Biotech,
Piscataway, NJ), and separated overnight by electrophoresis.
During the initial 3 h, the voltage was linearly increased from
300 to 3500 V, followed by 3 h at 3500 V, to reach the final
voltage of 5000 V. After separation in the first dimension, the strips
were equilibrated in 50 mM Tris-HCl, pH 8.4, 6 M urea, 30% glycerol,
2% SDS, and 2% dithioerythritol for 12 min. Thiol groups were
subsequently blocked with 2.5% iodoacetamide. Separation in the second
dimension was conducted using a vertical gradient slab gel with a
modified Laemmli-SDS discontinuous system (10% acrylamide-piperazine
diacrylyl gel) and run at 200 V for 
5 h. Gels were silver stained
according to a protocol compatible with MS (25).
Destaining, in-gel protein digestion, extraction, and purification
Each visible spot of the 2-D gel was cut and lyophilized in a
sterile Eppendorf tube. The silver stain was removed by covering the
gel piece with 30 mM K-ferricyanide and 100 mM Na-thiosulfate (1:1,
v/v), shaking for some minutes, and observing the destaining
(26). Each gel piece was then washed with water three
times, covered with 0.2 M ammonium bicarbonate, and incubated for at
least 20 min at room temperature (RT). The ammonium bicarbonate was
then removed, replaced with 100% acetonitrile, and washed three times.
Each gel piece was dried, and the digestion was started by adding
trypsin (Promega; 0.5 µg/gel piece) in 0.2 M ammonium bicarbonate and
kept on ice for 15–20 min. More buffer was added in small aliquots to
allow a slow uptake of the protease into the gel. The digestion was
conducted overnight at 37°C. The reaction was stopped by adding TFA
to a final concentration of 1%, and the sample was sonicated for 10
min. The supernatant was saved, 0.1% TFA/60% acetonitrile was added
to cover each gel piece, and the tube was incubated for at least 30 min
at 37°C. This extract was combined with the previous supernatant, and
extraction was repeated. A final extraction was performed, using 100%
acetonitrile. All extracts were then combined, and the volume was
reduced by vacuum centrifugation to about one-third of the original
volume. The samples were desalted by passing them though Zip-Tips (C18;
Millipore, Bedford, MA). The samples were lyophilized and resuspended
in 3 µl 0.1% TFA/H20. One microliter of the
sample was mixed (1:1, v/v) with matrix (a saturated solution of
-cyano-4-hydroxycinnamic acid in 50% acetonitrile/0.1% TFA) and
spotted on the target of the mass spectrometer.
Treatment of the bands excised from the Coomassie-stained gel was identical, except that the K-ferricyanide/Na-thiosulfate treatment was omitted.
Synthetic peptide digestion assays
A total of 4 nmol peptide was used in digestion assays with cytosol, its derived fractions, and purified peptidases. The digestions were allowed to proceed for the time indicated in the figures, and the reactions were stopped by adding 2% TFA. After lyophilization, the samples were analyzed by MS, as previously described (22). In digestion performed in the presence of peptidase inhibitors, the inhibitors were added to the cytosol and incubated for 15 min at RT prior to the addition of peptide. The concentration of butabindide (a kind gift of J.-C. Schwartz, Institut National de la Santé et de la Recherche Médicale Unité 109, Paris, France) was 5 µM, AAF-chloromethylketone (CMK) 50 µM, and puromycin 50 µM.
MALDI-TOF mass-spectroscopic analysis and database search
For protein identification, all analyses were performed using a Perseptive Biosystems MALDI-TOF Voyager DE-RP or a Voyager DE-STR mass spectrometer (Framingham, MA) operated in the delayed extraction and reflector mode. The search program ProFound, developed by The Rockefeller Mass Spectrometry Laboratory and New York University (New York, NY), was used for database searches (27). Peptides were selected in the mass range of 800-4000 Da. Spectra were calibrated using a matrix and tryptic autodigestion ion peaks as internal standards.
For regular peptide digestion assays, the settings of the instrument were as reported (22).
Cell fractionation
BB64-RCC (2 x 108 cells) were mechanically disrupted in hypotonic buffer, and the membranes were immediately equilibrated with 250 mM sucrose. Cell debris were pelleted at 13,000 x g for 10 min. The supernatant was subjected to high speed centrifugation (80,000 x g) for 45 min at 4°C. Incomplete cytosol (6 ml) was obtained by the removal of the proteasome from the clear supernatant, using immobilized anti-proteasome Ab MCP21. Membranes were resuspended in 2 ml RM buffer (50 mM HEPES, pH 7.2, 250 mM sucrose, 50 mM potassium acetate, 2 mM magnesium acetate) and pelleted at 80,000 x g for 45 min at 4°C. After repeating this procedure twice, the membranes were resuspended in 1 ml RM buffer containing 2% Triton X-114, maintained on ice for 10 min, and subjected to phase separation (28). This was performed by placing the samples at 37°C for 10 min and by subsequent centrifugation at 12,000 x g for 10 min at RT. The upper phase (fraction A), corresponding to the detergent-poor fraction and containing hydrophilic luminal proteins, was transferred into a new tube, and the remaining detergent-rich lower phase (fraction B), containing membrane-anchored proteins, was washed by adding fresh RM buffer lacking Triton X-114. The tube was placed on ice for 10 min and the phase separation was repeated. After two washing cycles, the volume of the detergent-rich phase was readjusted to the original volume (1 ml). Twenty microliters of incomplete cytosol, 4 µl fraction A, and 4 µl fraction B, which corresponds to the material obtained from an equivalent number of cells, were incubated with 4 nmol RU1 peptides for 20 min at 37°C. The reaction was stopped by adding 2% TFA, and the samples were analyzed by MS, as described above. Forty microliters of incomplete cytosol, 8 µl phase A, and 8 µl phase B were incubated with 100 µM AAF-AMC for 10 min at 37°C. Where indicated, the peptidase inhibitors butabindide (5 µM) and puromycin (50 µM) were added 15 min before the addition of the fluorogenic substrate.
Western blot analysis
Western blot analysis was performed according to standard procedures. A total of 20 µl cytosol, 4 µl fraction A, and 4 µl fraction B was separated on SDS-8% PAGE for the detection of TPP II and SDS-10% PAGE for the detection of PSA, and blotted onto nitrocellulose. The polyclonal chicken 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 PSA, a goat Ab anti-PSA (1:1000) Ab and a second Ab anti-goat peroxidase conjugate (1:3000) (Fluka) were used. The protein signals were revealed with ECL Western blotting detection reagent (Amersham Pharmacia Biotech).
Cloning of RU1 minigenes
The pEGFP-Ub vector is similar to the one described previously
(22), except that the green fluorescent protein (GFP)
moiety has been replaced by enhanced GFP (EGFP). Details are available
upon request. The cDNA fragments corresponding to the minimal
HLA-B*5101-restricted epitope RU134–42 and its
C-terminally extended version, RU134–47, were
obtained by annealing complementary oligonucleotides encoding the two
RU1-derived peptide fragments. The oligonucleotides were designed so as
to reconstitute a SacII site at the 5' end and an
AvaI site at the 3' end, and included a stop codon
immediately upstream of the AvaI site. Two additional
codons, specifying two Gly residues, were added at the 5' end of the
minigene so as to reconstitute the exact C terminus of the Ub moiety.
Upon annealing, the fragments were inserted between the
SacII/AvaI sites of pEGFP/Ub, resulting in
plasmid pEGFP/Ub-RU134–42 and
pEGFP/Ub-RU134–47. The same approach was used to
construct the plasmid
pEGFP/Ub
G75,76-RU134–42
coding for an uncleavable fusion
EGFP-Ub-RU134–42, except that the codons
specifying the Gly at the 3' end of Ub were omitted. The EGFP-Ub
plasmid control coded for EGFP-Ub-Melan-AMART1,
in which Melan-AMART1 is a melanoma-associated
protein of 118 aa.
Cell lines, transient transfections, and metabolic labeling
The human renal carcinoma BB64-RCC and the human embryonic kidney HEK293 and HEK293EBNA (EBV-encoded nuclear Ag) cell lines were maintained in DMEM medium (Invitrogen), supplemented with 10% FCS, antibiotics, and 20 mM Na HEPES, pH 7.3.
Transient transfections of the HEK293-EBNA cells were performed using
the Lipofectamine reagent (Invitrogen). Fifty thousand cells were
plated into flat-bottom microwells and transfected with 1.5 µl
Lipofectamine, 20 ng plasmid pcDNA3 containing the HLA-B*5101 cDNA, and
12.5 ng of either pcDNA3.1TOPO plasmid (Invitrogen) containing the RU1
full-length cDNA, pEGFP/Ub plasmid containing the cDNA-encoding
RU134–42, or pEGFP/Ub-encoding
RU134–47. A total of 100 ng pBJ1neo construct
encoding the herpes simplex-derived TAP inhibitor ICP47 molecule (a
kind gift of H. G. Rammensee, Tübingen, Germany) was added
to this mix in one-half of the wells. After 20 h, the transfected
cells were incubated with 104
anti-RU134–42/HLA-B51 CTL 381/84, along with
25 U/ml IL-2. The amount of TNF- secreted in the supernatant was
assessed 24 h later by ELISA (Endogen, Woburn, MA).
For metabolic labeling, 2 x 106 HEK293 cells were transfected with 4 µg plasmid using the Fugene reagent (Roche) and following the manufacturer’s protocol. Sixteen hours posttransfection, the cells were starved for 45 min in Met/Cys-free DMEM medium (ICN Biomedicals, Aurora, OH) at 37°C. The cells were metabolically labeled for 20 min at 37°C in fresh Met/Cys-free medium containing 150 µCi [35S]Met/Cys (Pro-mix; Amersham Pharmacia Biotech). Cells were washed once and lysed in 1 ml lysis buffer containing 1% Triton X-100 and 30 mM iodoacetamide to prevent postlysis deubiquitylation. Unsolubilized material was removed by centrifugation, and the supernatant was incubated with saturating amounts of the mAb anti-hemagglutinin (HA) epitope (Berkeley Antibody, Richmond, CA) and 20 µl protein G-Sepharose. The immunoprecipitates were washed and treated, as described earlier (22), subjected to SDS-12% PAGE, followed by autoradiography.
Treatment of target cells with inhibitors of Ag processing
BB64-RCC cells were acid treated as follows to discard peptides
from the surface HLA molecules. One million cells were incubated for
exactly 30 s at 37°C in 500 µl 300 mM glycine buffer (pH 2.5),
supplemented with 1% sterile BSA, and then washed several times with
culture medium. Eight thousand cells seeded in microplates were
incubated for 14 h with 50 µM lactacystin (Calbiochem, La Jolla,
CA) or 100 µM AAF-CMK in culture medium. Thereafter, some of the
cells were pulsed for 30 min with the RU1 antigenic peptide VPYGSFKHV
at a final concentration of 10 µM, washed, and incubated with 3000
CTL 381/84 and 25 U/ml IL-2. The ability of the treated cells to
stimulate the CTL was assessed by measuring the production of TNF by
the CTL after 20 h of incubation. The TNF ( and 
) content of
the supernatants was evaluated by testing their cytotoxic effect on
WEHI-164 clone 13 cells (29).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Earlier work, using the precursor peptide RU124–47, demonstrated that the exact C terminus of the antigenic peptide was directly produced by purified standard proteasome, but that the N terminus always carried additional 3 (RU131–42) and 5 (RU129–42) aa (16) (data not shown). This suggested that other peptidases might be necessary to trim the N-terminal extensions to the final nonamer RU134–42. We therefore sought to identify the cytosolic peptidases involved in the N-terminal editing of the RU134–42 epitope precursors. Because we were not able to determine the relative abundance of each of the two fragments produced by the proteasome due to their coelution from the HPLC columns (data not shown), we investigated the editing of both species.
We first focused our attention on the precursor
RU129–47, which carries an N-terminal extension
of 5 aa, and whose C-terminal processing was studied in our previous
work (Fig. 1
) (16). To
identify the peptidase(s) involved in the N-terminal trimming of this
precursor, we adopted the following strategy: proteasome-depleted
cytosol isolated from the renal carcinoma cell line BB64-RCC was
fractionated by ion-exchange chromatography. The presence of a
proteolytic activity was assayed by incubating an aliquot of each
fraction with the peptide RU129–47 and by
subsequent analysis by MS. The 5-aa extension at the C terminus of the
antigenic peptide sequence was included in the precursor so as to
monitor for the possibility that a peptidase other than the proteasome
may directly generate the CTL-defined epitope. No fraction was found to
generate the exact N terminus of the antigenic peptide. Rather,
fractions 10–14, eluting at 420–460 mM NaCl, contained an activity
that resulted in the trimming of the first 3 N-terminal aa (Fig. 2, A and B). No
proteolytic activity on the C-terminal extension of the antigenic
peptide was detected in any fraction (data not shown). Fractions 10–14
were pooled and separated by 2-D gel electrophoresis. After silver
staining of the gel, 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
ProFound (http://129.85.19.192/profound_bin/WebProFound.exe) and
used for peptide mass fingerprinting. Based on the pattern of its
tryptic fragments and its migrating properties in the 2-D gel, one spot
was unambiguously identified as TPP II. No other peptidases were
identified among the analyzed spots. TPP II is a very large
homomultimeric peptidase (molecular mass 5000–9000 kDa), with
subunit molecular mass 138 kDa, that removes tripeptides from the N
terminus of peptides and also displays endoproteolytic activities
(30, 31). Based on indirect evidence, TPP II has been
suggested to participate in the MHC class I Ag-processing pathway, as
it may partially compensate for the generation of MHC class I ligands,
in situations in which proteasomes are pharmacologically inactivated
(32). To confirm that the proteolytic activity producing
the fragment RU132–47 could be ascribed to TPP
II, we purified TPP II by immunoadsorption and assayed its activities
on the precursor RU129–47 (Fig. 2C).
After numerous unsuccessful attempts to purify active rTPP II from
prokaryotic as well as eukaryotic expression systems, we developed a
new purification scheme that yielded active TPP II from 5 x
107 cells. As demonstrated by the MS analysis,
the peak profile of the digested peptide, using immunoadsorbed TPP II,
was identical with the one detected after incubation of the same
precursor with cytosolic fraction 12 (Fig. 2, compare B and
C). Because the final N terminus of the antigenic peptide
was not detected, two possibilities were envisaged: either the
generation of the final N terminus was sequential and required another
peptidase for the removal of the last 2 aa, or the N-terminal
processing occurred entirely within membranes, which were absent from
this preparation.
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To address the first hypothesis, we investigated the possibility
that a second cytosolic peptidase might be responsible for the final
trimming of the N-terminal extension. To exclude the possibility of
contamination from peptidases located in subcellular compartments,
proteasome-depleted cytosol isolated from erythrocytes (these cells do
not contain internal membranes (33)) was subjected to
fractionation. The proteolytic activities of the fractions obtained
after separation on Q-Sepharose were tested using the fluorogenic
peptide AAF-AMC (Fig. 3A). Two
major peaks of activity were detected in fractions 29–33 and 38–45.
The activity of the first peak was able to digest the N terminus of
RU132–47, resulting in the enrichment of a
fragment corresponding to RU134–47, which
displays the exact N terminus of the antigenic peptide (Fig. 3A, inset). In light of this result, fraction 30,
the proteolytically most active fraction, was precipitated by TCA,
separated on SDS-PAGE, and stained by Coomassie blue. Each of the 12
visible protein bands was excised from the gel, digested by trypsin,
and further processed as above. The tryptic peptides obtained from a
protein band with apparent molecular mass 100 kDa led to the
identification of the peptidase puromycin-sensitive aminopeptidase
(PSA). This peptidase, originally found in brain tissues (see Ref.
34 and references therein), has recently been identified
as playing a role in the N-terminal trimming of another antigenic
peptide precursor (13). To ascertain the role of PSA in
the final N-terminal trimming of RU134–42, we
transfected and immunoadsorbed VSV-tagged PSA in the human embryonic
kidney cell line HEK293. Using the same technique developed for the
purification of active TPP II, immunoadsorbed and proteolytically
active PSA was used to digest the precursor peptide
RU132–47. Analysis of the digested material by
MS revealed that, after 2-h incubation at 37°C, the peak
corresponding to the original peptide RU132–47
had completely disappeared and a single peak corresponding to peptide
RU134–47 could be detected (Fig. 3B).
This fragment corresponds to a species displaying the exact N
terminus of the antigenic peptide (Fig. 1
). In contrast, the precursor
peptide RU129–47 was barely degraded by purified
PSA during the same time frame (Fig. 3B).
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TPP II and PSA can both edit the N terminus of peptide RU131–42 to its final size
Because neither TPP II nor PSA alone could efficiently trim
RU129–47 to the exact N terminus of the
CTL-defined epitope, we tested whether this was also the case for the
second N-terminally extended precursor produced by the proteasome,
RU131–42 (Fig. 1). Contrary to the first RU1
precursor, we used in this study a precursor that already displayed the
exact C terminus of the antigenic peptide. Using the same assay as
described above, we incubated the precursor peptide either with
immunoadsorbed TPP II (Fig. 4A) or PSA (Fig. 4B). Analysis of the digested products by MS revealed that
both peptidases were capable of trimming the N-terminal extension of
the precursor peptide to a fragment corresponding to the exact
antigenic peptide. The dichotomy between the processing of
RU129–47 and RU131–42 led
us to conclude that the sequential trimming of the first one by TPP II
and PSA may be caused by the presence of particular amino acids that
resist cleavage by PSA (see Discussion).
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To determine the contribution of the two identified cytosolic
peptidases to the N-terminal trimming of
RU134–42 precursors in unfractionated cytosol,
we performed a series of digestions of the various peptide precursors
in the presence or absence of specific TPP II and PSA inhibitors (Fig. 5). The peptides
RU129–47, RU131–42, and
RU132–47 (Fig. 1) were incubated with
proteasome-depleted cytosol for 10 min at 37°C (Fig. 5, A–C). The reaction was stopped by the addition of 2% TFA
and analyzed by MS. The same digestions were also performed in the
presence of butabindide, a specific TPP II inhibitor (Fig. 5, D–F) (35); AAF-CMK, an inhibitor of TPP II,
PSA, and bleomycin hydrolase (G–I) (13, 30);
and puromycin, a specific PSA inhibitor (J–L)
(34). In the case of RU129–47, a
fragment displaying the final N terminus of the antigenic peptide was
readily detectable after incubation with untreated cytosol (Fig. 5A). No such fragment could be detected when the peptide was
incubated with cytosol pretreated with butabindide or AAF-CMK (Fig. 5, D and G, respectively). Interestingly, not only
was the fragment with the exact N terminus absent, but the intermediate
corresponding to RU132–47 was not detectable
either. The result obtained after treatment with butabindide confirms
and extends the findings shown in Figs. 2 and 3, namely that the
processing of the N-terminal extension of
RU129–47 in unfractionated cytosol is a
sequential process that requires the activity of TPP II. Finally,
puromycin-treated cytosol led to an accumulation of the intermediate
RU132–47 by blocking the second proteolytic
event that normally leads to the generation of the final N terminus
(Fig. 5J).
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C) and was
insensitive to butabindide and only partially sensitive to AAF-CMK
(F and I, respectively). However, no digestion
was observed when the cytosol was pretreated with puromycin (Fig. 5L), confirming the essential role of PSA in the final
trimming of this intermediate. Finally, we analyzed the trimming of the
precursor RU131–42. As already predicted from
the result shown in Fig. 4, a fragment corresponding to the final size
of the antigenic peptide was produced both in untreated cytosol and in
cytosol treated with inhibitors (Fig. 5, B, E,
H, and K). We conclude that this precursor can be
edited simultaneously either by TPP II or PSA, but the contribution of
yet another peptidase on the editing of this particular peptide cannot
be ruled out, as peaks corresponding to fragments lacking 1 and 2
N-terminal aa could be detected in all conditions. The N-terminal processing of RU134–42 precursors occurs predominantly in the cytosol
Several reports have suggested that the N-terminal trimming
of antigenic peptides may occur within the ER. Although we presented
evidence that the processing of
RU134–42 precursors could occur in the
cytosol (Fig. 5), we nevertheless tested whether the trimming could
also take place within ER membranes. We purified membranes from the
BB64-RCC cell line, separated integral membrane proteins from soluble
luminal proteins by Triton X-114 extraction (see Materials and
Methods for details), and tested the proteolytic activity of each
of these fractions using the three peptide precursors. As before, we
could clearly detect proteolytic cleavage of all precursors in the
cytosolic fraction, resulting in the formation of a fragment displaying
the exact N terminus of the antigenic peptide (Fig. 6, A–C). Very little
proteolytic activity was detected in the fraction A, containing soluble
luminal proteins (Fig. 6, D–F), and no activity was
detected in the fraction B, containing membrane-embedded proteins (Fig. 6, G–I). Proteolytic activity present in the three
fractions was also independently monitored by the release of AMC from
the fluorogenic tripeptide AAF-AMC (Fig. 6J). The cytosolic
extract was proteolytically active against AAF-AMC, and these
activities could be partially blocked either by butabindide or
puromycin, indicating that: 1) TPP II and PSA are present in this
fraction, and 2) other peptidases are also active against this
fluorogenic peptide. The presence of the two peptidases was further
confirmed by Western blot analysis (Fig. 6K). Some
proteolytic activity could also be detected in fraction A. However,
this activity was totally resistant to puromycin (confirming the
absence of PSA from this fraction), but was completely blocked by
butabindide, indicating the presence of contaminating TPP II in this
fraction. This was independently confirmed by Western blot analysis
using an anti-TPP II Ab (Fig. 6K). The presence of TPP
II could be responsible for the small peak of
RU132–47 and RU132–42
detected in D and F, respectively. The presence
of TPP II in fraction A could be explained by the fact that a fraction
of this very large peptidase (5–9 Md) cosediments with
microsomal membranes. Indeed, we found that a 60-min centrifugation at
350,000 x g efficiently sediments most of the TPP II
present in the cell lysate (data not shown).
|
, B,
E, H, and K, peaks corresponding to
fragments missing 1or 2 N-terminal aa could also be observed in
digested material obtained from fraction A (Fig. 6F). This
proteolytic activity is insensitive to butabindide, puromycin, and
AAF-CMK (Fig. 5). It is noteworthy that both other precursors do not
seem to be substrate of this peptidase. We do not have any information
on this peptidase at the moment. The presentation of RU134–42/HLA-B51 is blocked by lactacystin and AAF-CMK
In an attempt to correlate our results obtained in an acellular
system with the cellular pathway leading to the presentation of
RU134–42 by HLA-B51, we treated BB64-RCC with
specific inhibitors and tested their recognition by CTL. Cells treated
with the proteasome inhibitor lactacystin were poorly recognized by
specific CTL, as indicated by the low level of TNF produced by the CTL
clone (Fig. 7). Recognition was partially
restored when the antigenic peptide was added exogenously. In
comparison, untreated cells were efficiently recognized by the same CTL
clone, confirming that the proteasome played an essential role in the
presentation of this CTL-defined epitope. We also tested whether the
recognition of RU134–42/HLA-B51 was influenced
by the presence of the inhibitor AAF-CMK. As with lactacystin, cells
treated with AAF-CMK were poorly recognized by specific CTL. Again,
exogenously added peptide led to a partial restoration of the
recognition of BB64-RCC cells. The lack of recognition resulting from
the AAF-CMK treatment could not be ascribed to an inhibition of
proteasome because, as tested by us and reported by others, proteasomal
activities were not influenced by AAF-CMK (13, 30) (data
not shown). In our hands, 100 µM lactacystin did not inhibit 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 generate the antigenic peptide. We were unable to test the effect of
butabindide on the presentation of RU134–42
because this drug does not cross the cell membrane (data not shown).
Puromycin could not be tested either, due to its excessive cell
toxicity (data not shown).
|
The antigenic peptide RU134–42 contains a
subaminoterminal P (Fig. 1) and is thus predicted to be poorly
transported by TAP in its final form. However, we have shown that the
production of the final N terminus can occur in the cytosol. To test
whether the N-terminally trimmed antigenic peptide can be transported
into the ER, the human embryonic kidney cells HEK293 were
transiently transfected with the cDNA-encoding HLA-B51 and either the
RU1 cDNA or two minigenes coding for, respectively, peptide
RU134–47, displaying the exact N terminus, but
carrying a C-terminal extension, and RU134–42,
the minimal HLA-B51-restricted 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 the tripartite linear
fusion protein EGFP-ubiquitin (Ub) minigene. The EGFP-Ub moiety is
cotranslationally cleaved after the last residue of Ub by the cytosolic
Ub peptidase, thereby liberating the peptides in the cytosol (37, 38). In addition, this gene arrangement also allows the
expression of minigenes without the need of the initiation codon for
methionine. As shown in Fig. 8A, cells transfected with the
three RU1 constructs were recognized by the specific CTL. It is
noteworthy that the cells transfected with the vector encoding the
minimal antigenic peptide sequence were more efficiently recognized by
the CTL, supporting the notion that the proteasomal processing may
limit the efficiency of Ag presentation. More efficient recognition of
DNA-encoded RU134–42 occurred when cells were
transfected with plasmid concentrations ranging from 1.25 to 50 ng
(data not shown). To confirm that the antigenic peptide was transported
by the TAP complex in our experimental conditions, the gene encoding
the natural TAP inhibitor ICP47 from herpes simplex was cotransfected
with the plasmids described above (39). As expected, these
cells were not recognized anymore, confirming the necessity of
functional TAP for the recognition of target cells (Fig. 8A). The slightly reduced inhibition of the recognition of
cells expressing the minimal epitope (RU134–42)
in presence of ICP47 could be explained by the incomplete effects of
ICP47 on TAP, a TAP-independent peptide transport, or the fact that a
small percentage of the peptide may be released into the extracellular
medium and recaptured by HLA-B51 molecules at the cell surface. In
other experiments, the effect resulting from the expression of ICP47
was confirmed to be specific, and not due to dilution by the additional
plasmid DNA transfected (data not shown). We conclude from these
experiments that the antigenic peptide RU134–42,
carrying P at position 2, can be translocated across the ER membrane in
its final form via the TAP transporters. We could exclude that the
fusion GFP-Ub could be cleaved within the ER because it lacks an
ER-targeting signal sequence and because the Ub-specific peptidase is
absent from the ER (38) (F. Lévy, unpublished
data).
|
G75,76-RU134–42,
which lacks the two C-terminal Gly residues of Ub, thereby producing an
uncleavable form of Ub. Indeed, the C-terminal Gly have been shown to
be essential for the Ub peptidase-mediated cleavage of Ub
(40). Both constructs contained a sequence derived from
the influenza A HA between the C terminus of EGFP and the N terminus of
Ub, which can be recognized by a specific Ab (22). Cells
were transfected with both plasmids, metabolically labeled with
[35S]Met/Cys, and lysed in the presence of
alkylating agent to prevent postlysis deubiquitylation, and the cleared
lysate was immunoprecipitated using the anti-HA mAb linked to
protein G-Sepharose. The length of the RU1 minigenes was selected so as
to maximize the migration differences after separation on SDS-PAGE. As
shown in Fig. 8B, the SDS mobility of
EGFP-Ub-RU134–47 carrying the wild-type Ub
moiety was indistinguishable from EGFP-Ub derived from a plasmid
encoding a melanoma-associated protein fused to EGFP-Ub. In contrast,
EGFP-UbG75,76-RU134–42
migrated with a slightly slower mobility. This result indicated that
the cleavage at the Ub minigene junction had occurred in the wild-type
construct and produced a polypeptide that differs from the mutant
construct by 7 aa. A very faint band with slower mobility could be
observed for the nonmutated construct, but its precise identity could
not be determined because a band of identical mobility was also visible
in the construct carrying the mutated Ub and could be sometimes
detected in the EGFP-Ub control plasmid. Even though the vast majority
of EGFP-Ub-RU134–42 molecules was cleaved, the
possibility that a small fraction of
EGFP-Ub-RU134–42 may not be cleaved by
Ub-specific proteases and produces an amino-terminally extended
antigenic peptide precursor cannot be completely ruled out.
Nevertheless, the data presented in this work support the notion that
the final processing of RU134–42 can take place
in the cytosol. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The N-terminal trimming peptidases identified to date share the
properties of having broad specificities, of being redundant, and of
trimming antigenic peptide precursors without detectable accumulation
of a species with the correct N terminus. We were therefore surprised
that the generation and the transient accumulation of the final N
terminus of RU134–42 from
RU129–47 required the sequential action of two
distinct peptidases. The two identified peptidases, TPP II and PSA,
have already been implicated, in different experimental conditions, in
the MHC class I Ag-processing pathway. The first one, TPP II, was
identified using cells that had been adapted to a proteasome inhibitor
(32). This treatment induced the overexpression of TPP II,
and indirect evidences, such as cell surface expression of MHC class I
and HPLC profile of peptides eluted from MHC class I molecules,
suggested that TPP II may compensate for the lack of proteasome
activity (32, 41). However, the exact contribution of TPP
II to this process remains to be elucidated, as the activities of the
proteasome may not be completely blocked in those cells
(42). Another study demonstrated that TPP II did not only
remove tripeptides from the N terminus of peptides, but also had some
endoproteolytic activities, which could potentially produce antigenic
peptides (30). Of note is that in this study we did not
observe any other endoproteolytic activity of human TPP II than the one
removing tripeptides from free N termini (data not shown). However, in
none of the two cases mentioned above was TPP II directly identified in
the trimming of a specific antigenic peptide precursor. Contrary to the
role of TPP II in the production of antigenic peptides, the
contribution of PSA to the processing of a CTL-defined epitope was
recently revealed by an experimental approach similar to the one
described in this work (13). In that approach, PSA was
shown to degrade N-terminally extended precursors of the antigenic
peptide VSV nuclear protein52–59 and to
generate, among many other fragments, a peptide with the final N
terminus. Using the precursor peptide RU129–47,
we showed that the production of the final N terminus of the antigenic
peptide was an ordered process, which required the sequential
activities of the peptidases TPP II and PSA (Figs. 3, 4, and 6). This
was also the case in a cytosolic extract, which contained other active
peptidases (Figs. 6 and 7J). A molecular explanation for
this resides probably in the specificity of the individual peptidases.
It appears that neither TPP II nor PSA can cleave a peptide bond before
or after Pro (P). In addition, PSA does not cleave efficiently peptide
bonds involving a Gly (G) residue (34). Because the
sequence of the precursor peptide is TGSTAVP... , in which VP
corresponds to the first 2 aa of the CTL-defined epitope (Fig. 1), this
may explain why TPP II has to remove the first 3 aa (it cannot cleave
further because this would involve the residue P) before PSA, which
does not cleave the full-length precursor because of the amino acid G,
can trim the last 2 aa. PSA will then stop at the exact N terminus of
the antigenic peptide because of the residue P. Such a process may be
specific for antigenic peptides carrying the residue P at position 2,
as is the case for certain ligands of the HLA-B7, HLA-B8, HLA-B15,
HLA-B51, and other class I molecules. However, the fact that the same
peptidase (PSA) has been identified using two different peptide
precursors may not be coincidental, and leads us to postulate that a
limited number of peptidases will be responsible for the final editing
of MHC class I ligands. At present, we do not know whether this
sequence of action is identical in all cell types nor in situations in
which the expression of other peptidases is induced, as is the case for
leucyl aminopeptidase induced by IFN-
(15).
The production of the final antigenic peptide from the N-terminally extended precursor RU131–42 could be achieved by TPP II and PSA in a redundant fashion. This result is in agreement with the findings reported on the generation of the N-terminal end of VSV nuclear protein52–59, namely that the final N terminus of this antigenic peptide could be obtained by two redundant processes, mediated either by PSA or, in that case, by bleomycin hydrolase (13). The likely explanation for the different processing of RU131–42 and RU129–47 resides in the length and the sequence of the N-terminal amino acid extension. Therefore, the choice of peptidase(s) responsible for the postproteasomal trimming of antigenic peptide precursors may be dictated by the nature of the N-terminal extension produced by the proteasome. We have analyzed the processing of several antigenic peptide precursors in vitro and have noticed that the proteasome can, in some instances, directly generate the exact antigenic peptide (our unpublished data). However, this species is frequently accompanied by the presence of peptides that carry additional N-terminal amino acids. It will be interesting to study whether the final antigenic peptide produced within a cell will be more efficiently loaded onto MHC class I molecules than the precursors requiring further N-terminal processing.
N-terminal trimming in the ER has also been described (5).
However, because no specific peptidase has been identified at the
molecular level, it is difficult to speculate on the role of this ER
trimming. The fact that most peptides isolated from the HLA-A2
molecules of the TAP-deficient cell line T2 are considerably longer in
size than those isolated from normal cells (43) indicates
that if aminopeptidases are active in the ER, they may be very specific
or play a minor role in the processing of antigenic peptides derived
from signal sequences released by the signal peptidase in the ER. In
this study, we directly searched for membrane-embedded or luminal
peptidases that would mediate the final trimming of our precursor (Fig. 7). Although we could detect a proteolytic activity other than the one
mediated by TPP II in our membrane preparation, it was only acting on
the precursor RU131–42 and was also found in the
cytosol. Moreover, this activity was not inhibited by AAF-CMK even
though this drug efficiently blocked the CTL recognition of APCs,
suggesting that this peptidase is not participating to the final
processing of RU134–42. One reason for this is
that this peptidase is localized in another subcellular compartment,
which contaminated our microsomal preparation. Alternatively, the
product RU131–42 detected in our in vitro
digestion assay may constitute only a minor species that is not
produced intracellularly. Irrespective of the role of this peptidase in
this process, our data suggest that the antigenic peptide
RU134–42 is made in the cytosol and can be
transported as such across the ER membrane.
After prolonged incubation of the various RU134–42 precursors in cytosol, the peptides were eventually completely degraded (data not shown). This may be explained by the fact that our in vitro digestion assay does not contain any membrane, which, in cells, can offer a physical barrier against the attack of other cytosolic peptidases. However, the mechanism by which antigenic peptides produced in their final form in the cytosol reach the TAP transporters has not been elucidated. Interestingly, a membrane-associated protein of 100 kDa, p100, has been identified, which binds peptides, possibly transiently, on the cytoplasmic side of the ER membrane (44). The association between this protein and the membrane appears to be mediated by an unknown protein. Because the m.w., pI, and peptide interaction properties of p100 are similar to those of PSA, it is tempting to speculate that p100 could be PSA. If this were the case, it would provide a mean to simultaneously edit the N terminus of longer peptide precursors and increase the chance of having the final antigenic peptide transported by the TAP1/2 complex. Experiments aimed at elucidating the intracellular localization of PSA are underway.
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Acknowledgments |
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Footnotes |
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2 F.L. and L.B. contributed equally to this study.
3 Address correspondence and reprint requests to Dr. Frédéric Lévy, Ludwig Institute for Cancer Research, Ch. des Boveresses 155, CH-1066 Epalinges, Switzerland. E-mail address: Frederic.Levy{at}isrec.unil.ch
4 Current address: Institute of Experimental Immunology, Department of Pathology, University of Zürich, CH-8091 Zurich, Switzerland.
5 Abbreviations used in this paper: ER, endoplasmic reticulum; AMC, 7-amido-4-methylcoumarin; CMK, chloromethylketone; 2-D, two-dimensional; EBNA, EBV-encoded nuclear Ag; EGFP, enhanced GFP; GFP, green fluorescent protein; HA, hemagglutinin; MALDI-TOF, matrix-assisted laser desorption ionization-time of flight; MS, mass spectrometry; PSA, puromycin-sensitive aminopeptidase; RCC, renal cell carcinoma; RT, room temperature; TFA, trifluoroacetic acid; TPP II, tripeptidyl peptidase II; Ub, ubiquitin; VSV, vesicular stomatitis virus.
Received for publication May 1, 2002. Accepted for publication August 7, 2002.
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