From the Department of Cell Biology, Harvard Medical School,
Boston, Massachusetts 02115 and the Department of
Pathology, University of Massachusetts Medical Center,
Worchester, Massachusetts 01655
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ABSTRACT |
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Most antigenic peptides presented on major
histocompatibility complex class I molecules are generated during
protein breakdown by proteasomes, whose specificity is altered by
interferon- (IFN-
). When extended versions of the
ovalbumin-derived epitope SIINFEKL are expressed in vivo,
the correct C terminus is generated by proteasomal cleavage, but
distinct cytosolic protease(s) generate its N terminus. To identify the
other protease(s) involved in antigen processing, we incubated soluble
extracts of HeLa cells with the 11-mer QLESIINFEKL, which
in vivo is processed to the antigenic 8-mer (SIINFEKL) by a
proteasome-independent pathway. This 11-mer was converted to the 9-mer
by sequential removal of the N-terminal residues, but surprisingly the
extract showed little or no endopeptidase or carboxypeptidase activity
against this precursor. After treatment of cells with IFN-
, this
N-terminal trimming was severalfold faster and proceeded to the
antigenic 8-mer. The IFN-treated cells also showed greater
aminopeptidase activity against many model fluorogenic substrates. Upon
extract fractionation, three bestatin-sensitive aminopeptidase peaks
were detected. One was induced by IFN-
and was identified
immunologically as leucine aminopeptidase (LAP). Purified LAP, like the
extracts of IFN-
-treated cells, processed the 11-mer peptide to
SIINFEKL. Thus, IFN-
not only promotes proteasomal cleavages that
determine the C termini of antigenic peptides, but also can stimulate
formation of their N termini by inducing LAP. This enzyme appears to
catalyze the trimming of the N terminus of this and presumably other
proteasome-derived precursors. Thus, susceptibility to LAP may be an
important influence on the generation on immunodominant epitopes.
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INTRODUCTION |
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Mammalian cells present to the immune system small fragments of
intracellular proteins in the form of 8-10-residue peptides bound to
surface major histocompatibility complex
(MHC)1 class I molecules.
This process allows cytotoxic T cells to screen for intracellular
pathogens (e.g. viruses) and for transformed cells. The
great majority of these antigenic peptides is generated during the
course of protein degradation by 20 S or 26 S proteasomes (1), which
catalyze the breakdown of most cell proteins. Nearly all of the peptide
products of the proteasomes undergo rapid proteolytic destruction to
amino acids. However, some oligopeptides, perhaps after further
proteolytic processing, are transported from the cytosol into the
endoplasmic reticulum (ER), where they bind to MHC class I molecules,
and this complex is transported to the cell surface (2-4). Many lines
of evidence have indicated that the proteasome is essential for the
generation of most antigenic peptides. 1) Proteasome inhibitors, such
as peptide aldehydes (5, 6) and lactacystin -lactone (7, 8), prevent
the generation of most class I-presented peptides and the cytotoxic T-cell response. 2) Antigen presentation from certain proteins requires
their conjugation to ubiquitin, which leads to rapid breakdown by
the 26 S proteasome (9, 10). 3) The cytokine IFN-
, which stimulates
many steps in antigen presentation, induces the expression of three
special
-subunits (LMP2, LMP7, and MECL1). Their incorporation into
the 20 S proteasome modifies its peptidase activities and thus appears
to increase the generation of peptides with hydrophobic and basic C
termini (11-14). Such peptides are selectively transported into the ER
(15) and bind preferentially to MHC class I molecules (16). These
adaptations are clearly important in vivo, since deletion of
LMP2 (17) or LMP7 (18) genes in mice leads to defects in their ability
to generate cytotoxic T-cell responses. 4) IFN-
also induces
an 11 S complex, PA28, which stimulates the proteasome's
peptidase activity (19, 20) and thus may also promote antigen
presentation (21).
One important aspect of this process that is poorly understood concerns the exact fate of peptides released by the proteasome. The peptide-binding cleft of the MHC class I molecule binds strongly only peptides of 8-10 residues in length (22, 23). The sizes of the peptides containing antigenic epitopes that are released by the mammalian proteasomes are unknown. Recently, proteasomes from the achaeon, Thermoplasma acidophilum, have been shown to cleave proteins to peptides ranging in length from 3 to 25 amino acids. Even though the eukaryotic proteasome has many fewer active sites, and these sites differ in cleavage specificity (1), it generates peptides during protein breakdown whose size distribution resembles that of the achaeal proteasome (25).2 With both types of proteasomes, 10-15% of the peptide products are of the correct length for MHC class I binding (24). It thus remains unclear whether most presented peptides are produced directly by the proteasome, as suggested by some workers (27, 28), or whether additional proteolytic steps are necessary to generate the final 8-10-mers (29) (see below). Also, the enzymatic steps in epitope production may differ for different proteins. One of the epitopes studied more extensively is the ovalbumin-derived, H2-Kb-presented epitope, SIINFEKL. When SDS-activated 20 S proteasomes were incubated with ovalbumin (25) or with fragments of ovalbumin (27) for prolonged periods, SIINFEKL was generated, but also some N-terminal-extended versions of this peptide were produced. However, the physiological relevance of such experiments is unclear; for example, it remains questionable whether the products released by these 20 S particles under these artificial conditions are the same as those generated from ubiquitinated proteins by the 26 S proteasomes in vivo.2
Craiu and co-workers (29) showed that if longer peptides that contain
this antigenic sequence are injected into cells or expressed from
minigenes, these peptides could be proteolytically trimmed to SIINFEKL
and be presented on surface MHC class I molecules. Interestingly,
generation of this antigenic peptide from ovalbumin with C-terminal
extensions of SIINFEKL of 1-15 amino acids was completely blocked by
treatment of the cells with the proteasome inhibitor, -lactone (29).
Although the proteasome thus seems to play a critical role in the
generation of the C terminus of this antigenic peptide, this particle
was not necessary for the cleavages that define their N termini. When
peptides containing 2-25 additional residues on the N terminus of
SIINFEKL were expressed or injected into cells, SIINFEKL was presented
by MHC class I molecules, and this process was not affected by a
proteasome inhibitor. The 20 S proteasome, although it contains several
endopeptidase activities, lacks aminopeptidase activity. Thus, some
other proteolytic enzyme(s) must be generating the correct N terminus
of this (and presumably of other) antigenic peptides. Several findings
indicate that the major peptidase(s) active in this N-terminal trimming are located in the cytosol (29). For example, a SIINFEKL-containing precursor with 25 additional N-terminal residues was efficiently trimmed to the presented octapeptide by a non-proteasomal mechanism. However, peptides longer than 16 residues are quite poor substrates for
the TAP transporter on the ER (30). Therefore, most of the additional
N-terminal residues must have been cleaved off in the cytosol before
uptake into the ER for MHC class I binding, although some
exopeptidase(s) clearly capable of trimming antigenic peptides are also
found in the ER (31, 32).
A number of cytosolic proteases degrade preferentially oligopeptides
and therefore may function in the trimming or further degradation of
proteasome products, including the heterogenous group of
aminopeptidases (33-35). Unidentified bestatin-sensitive aminopeptidases have been shown to catalyze the final steps in the
ATP-dependent proteolytic pathway in the conversion of
small peptides to amino acids (33, 36). The present experiments were
undertaken to investigate which cytosolic proteases may be involved in
the post-proteasomal processing of class I-presented peptides, and
specifically in the cytosolic trimming of N-terminal-extended peptides
to SIINFEKL. In addition, we have tested whether IFN- treatment,
which promotes many important steps in antigen presentation, also
stimulates the processing of such peptides.
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EXPERIMENTAL PROCEDURES |
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Peptides and Reagents--
Peptides with the sequences SIINFEKL,
ESIINFEKL, LESIINFEKL, and
QLESIINFEKL were synthesized by Macromolecular Resources (Colorado State University, Fort Collins, CO) and were over 90% pure
(by HPLC analysis). The peptides were dissolved at 10 mg/ml in dimethyl
sulfoxide and stored at 80 °C. Purified porcine kidney leucine
aminopeptidase and tissue carboxypeptidase A were obtained from Sigma.
Bestatin was purchased from Sigma and the proteasome inhibitor, MG132
(Cbz-LLLal), was kindly provided by ProScript Inc. (Cambridge, MA). The
10-mer QLESIINFEK was prepared by incubation of the 11-mer
with carboxypeptidase A, and the product was isolated by HPLC.
Cell Lines--
The human cervical carcinoma cell line, HeLa S3,
and the human macrophage cell line, U937, were obtained from the
American Type Culture Collection and grown in Dulbecco's modified
Eagle's medium (Irving Scientific, Santa Ana, CA) supplemented with
10% fetal calf serum and antibiotics. HeLa and U937 cells were treated for 5 days with 1500 and 3000 units/ml of human recombinant IFN- (a
kind gift from Biogen, Cambridge, MA).
Preparation of Soluble Extracts--
Cells were homogenized in a
Dounce homogenizer and by vortexing with glass beads in 50 mM Tris-HCl, 5 mM MgCl2, 2 mM ATP, 1 mM dithiothreitol, and 250 mM sucrose, pH 7.4. Cytosolic extracts were prepared by
centrifugation of the homogenates for 20 min at 10,000 × g and 1 h at 100,000 × g, and
proteasomes were removed by an additional 6 h centrifugation at
100,000 × g. All extracts were stored at 80 °C
until use. For some experiments, the residual proteasomes in the
extracts were inactivated by incubation with 100 µM MG132
for 15 min at room temperature.
Peptidase Assay-- Aminopeptidase activity was analyzed using fluorogenic substrates of the amino acid-AMC type (Bachem, King of Prussia, PA). Substrates containing 19 different N-terminal amino acids (except tryptophan) were used at a concentration of 200 µM of each in a 1-ml volume of 50 mM Tris-HCl, 5 mM MgCl2, pH 8.5. Substrate hydrolysis was determined using 10 µg of cytosolic proteins from HeLa or U937 cells, as measured using the Coomassie kit from Pierce (Rockford, IL). Samples were incubated for 75 min at 37 °C, and the reaction was stopped by adding 1 µl of 10% SDS, and then fluorescence was measured at an excitation wavelength of 380 nm and an emission wavelength of 440 nm in a SLM-AMINCO spectrometer (Rochester, NY). Analysis of fractionated extracts was carried out with 50 µl of each fraction in 500 µl of 50 mM Tris-HCl, 5 mM MgCl2, pH 8.5, stopped after 75 min incubation at 37 °C with 500 µl of 2% SDS, and analyzed as described above.
High Performance Liquid Chromatography (HPLC) Analysis of Peptides-- 5 nmol of the synthetic peptide QLESIINFEKL was incubated with 10 µg of extract from HeLa cells or with 25 µg of extract from U937 cells in 100 µl of 50 mM Tris-HCl and 5 mM MgCl2, pH 8.5, for various times at 37 °C. The reaction was terminated by addition of 100 µl of 20% trichloroacetic acid followed by 15 min incubation on ice, and the precipitated protein was removed by centrifugation for 15 min at 14,000 rpm. The peptide-containing supernatant was subjected to reverse-phase HPLC on a 4.6 × 250-mm Macrosphere 300A C8-column (Alltech, Deerfield, IL) at 40 °C in 10 mM phosphate buffer, pH 6.8, with a flow rate of 0.75 ml/min. Elution was performed with a 25-min linear gradient from 15 to 40% acetonitrile. The eluted peptides were detected by measuring their absorbance at 214 nm. The relative concentrations of each eluted peptide were calculated by integration of the areas under the peaks and are given in arbitrary units.
Fractionation of Soluble Extract by Ion Exchange Chromatography-- Fractionation of 2 mg of U937 and 0.5 mg of HeLa cell extracts was performed by ion-exchange chromatography in 50 mM Tris-HCl buffer, pH 8.5, on a 1-ml MonoQ 5/5 column (Pharmacia, Upsala, Sweden). Bound proteins were eluted with a 20-min linear salt gradient from 0 to 0.5 M sodium chloride and with a flow rate of 1 ml/min. The eluted peptides were measured at 280 nm, and fractions of 0.5 ml were collected for further analysis.
Electrophoretic Methods and Immunoblot Analysis-- The identification and measurements of LAP in soluble extracts were done by immunoblot analysis. 30 µg of crude extract or 20 µl of each sample from fractionated extracts were separated on a 12.5% SDS-polyacrylamide gel, and the proteins were transferred onto an Immobilon P membrane (Millipore). The filters were blocked for 1 h at room temperature with 0.5% milk powder in phosphate-buffered saline and incubated overnight at 4 °C with a rabbit antiserum against bovine lens LAP (kindly provided by A. Taylor, Tufts University, Boston, MA). Bound antibodies were detected with 125iodinated-protein A (NEN Life Science Products) and visualized and quantified with a PhosphorImager (Molecular Dynamics).
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RESULTS |
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The Precursor QLESIINFEKL Is Processed to SIINFEKL in Cell Extracts-- Because in vivo studies (29) indicated that cytosolic protease(s) distinct from the proteasome generate the N terminus of the MHC class I-presented SIINFEKL from longer precursors, such as QLESIINFEKL, we tested whether a similar processing of this precursor occurs in soluble extracts of HeLa cells. The processing reaction was followed until the antigenic 8-mer was generated (on the assumption that once the 8-mer is generated in vivo, it would be efficiently transported into the ER and would bind to MHC class I molecules where it would be protected from further proteolysis). In principle, conversion of the ovalbumin-derived 11-mer to SIINFEKL can occur by a single endoproteolytic cleavage, by sequential removal of the N-terminal residues (generating the 10-mer and then the 9-mer), or by some mixture of endo- and exopeptidase reactions. To follow these reactions, an assay using reverse-phase HPLC was developed, which allowed resolution of the 11-mer (QLESIINFEKL), 10-mer (LESIINFEKL), 10-mer (QLESIINFEK), 9-mer (ESIINFEKL), and 8-mer (SIINFEKL) peptides. The 11-mer peptide QLESIINFEKL was incubated with the 100,000 × g supernatant from HeLa cells at 37 °C for various periods of time. To prevent proteasomal activity, the extracts were depleted of proteasomes by prolonged ultracentrifugation, and any residual activity was blocked by the addition of the inhibitor, MG132 (37), which blocks proteasome function, but does not inhibit aminopeptidases or carboxypeptidases. The reaction was stopped by addition of trichloroacetic acid, and the peptide-containing supernatant was fractionated by HPLC. Determination of the retention times of standard peptides allowed us to test whether the same peptides were generated in the extracts, and their relative concentrations were determined by integration of peak areas.
Initially, the 11-mer was the only peptide peak detected at 214 nm in the HeLa extract (Fig. 1). The lack of endogenous peptides in these undialyzed extracts confirms prior observations that the concentration of free peptides in the mammalian cytosol is very low (38). Extract concentrations (100 µg/ml) were studied that allowed easy measurement of the disappearance of the added 11-mer in a few hours. In the extracts, the amount of the added 11-mer decreased at a linear rate, and by 3 h, nearly 50% had disappeared (Fig. 2). After 30 min, additional peaks could be detected, which correspond to the 10-mer and 9-mer. These two peptides increased with time and reached similar maximal levels at 2 and 3 h. Only a very small peak corresponding to the C-terminal truncated 10-mer (QLESIINFEK) was detected after addition of the 11-mer to the HeLa extracts (data not shown). In these extracts, the amount of this C-terminal truncated peptide was always much less than the amounts of the N-terminal truncated peptides. These findings and inhibitor studies (see below) all indicated that in these preparations peptide processing occurred primarily by aminopeptidase(s), with little or no carboxypeptidase activity or endoproteolytic generation of the 9- or 8-mer.
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IFN- Increases Cellular Aminopeptidase Activities--
To
further characterize the aminopeptidase(s) induced by IFN-
, we used
a variety of fluorogenic aminopeptidase substrates. Initial experiments
with control and IFN-treated HeLa and U937 extracts using Leu-AMC as
substrate indicated that its hydrolysis occurred at a linear rate for
up to 75 min. The rate of this reaction was directly proportional to
the substrate concentration (which ranged from 50 to 600 µM) and to the amounts of extract protein (which ranged
from 10 to 50 µg) (data not shown). In extracts from IFN-treated
cells, the rate of Leu-AMC hydrolysis was consistently 2-3-fold higher
than in extracts from controls. We then compared the activity of the
control and IFN-treated cell extracts against 18 other amino acid-AMC
substrates (Table I). The substrates fell
into two groups, according to their rates of hydrolysis. In control
extract, the amino acid-AMC substrates containing Leu, Lys, Met, Cys,
Phe, Arg, Ala, Pro, and Tyr were hydrolyzed at least 2-20-fold faster
than those containing Thr, Gln, Glu, Asn, His, Val, Ser, Ile, Asp, and
Gly.
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The IFN--inducible Aminopeptidase(s)--
To identify the
aminopeptidase(s) which are induced in the cytosolic extracts by
IFN-
, extracts from control and IFN-treated HeLa cells were
fractionated by ion exchange chromatography, and the fractions were
analyzed for aminopeptidase activity against Leu-, Cys-, Lys-, and
Gln-AMC. These substrates represent ones that were hydrolyzed rapidly
(Leu-, Cys-, and Lys-AMC) or slowly (Gln-AMC) and ones whose hydrolysis
was unaffected (Lys-AMC) or stimulated by IFN-
(Leu-, Cys-, and
Gln-AMC). In both control and IFN-treated extracts, the cleavage of
Leu-AMC was found in two distinct peaks, which were eluted at 0.2 M (peak A) and at 0.35 M (peak
B) NaCl (Fig. 4). The peptidase
activity in peak A was 5-fold greater after IFN treatment than in the
controls. In contrast, IFN-
treatment had no stimulatory effect on
the activity in peak B. The hydrolysis of Cys-AMC was observed in the
same two peaks, and again this activity in peak A increased 2.4-fold
after IFN treatment, while peak B did not change significantly.
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The IFN-inducible Protease Is Leucine Aminopeptidase
(LAP)--
Because the IFN-induced aminopeptidase peak preferentially
hydrolyzed Leu-AMC and was sensitive to bestatin, it seemed possible that this enzyme corresponded to LAP. In fact, Harris et al.
(39) had found that one of the genes induced by IFN- in human
fibroblasts and certain carcinoma and melanoma lines codes for LAP. To
test whether peak A corresponded to LAP, we used a polyclonal rabbit antiserum against bovine lens LAP (kindly provided by A. Taylor, Tufts
University School of Medicine, Boston, MA) in an immunoblot analysis on
control and IFN-treated extracts from HeLa and U937 cells. With this
serum, we were able to detect cross-reacting bands in the extracts from
IFN-treated HeLa and U937 cells that had a similar molecular weight as
porcine kidney LAP (pkLAP) (Fig. 5A). Only a faint band could
be detected in the control extracts from these cells. Quantification of
the Western blot using 125I-protein A showed a 6-fold
induction of LAP in HeLa cells and a 14-fold induction in U937 cells
after treatment with IFN-
.
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DISCUSSION |
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Although the great majority of the peptides released by
proteasomes during protein breakdown are rapidly degraded to amino acids, a fraction is used for antigen presentation (6, 7). Depending on
the nature of the products produced, these peptides may either be
presented without further modification (25, 27) or may have to be
further trimmed by other proteases before presentation (29). In the
present study, we found that cytosolic extracts of HeLa cells, like
intact cells, can convert extended forms of the immunodominant
ovalbumin epitope, SIINFEKL, to the presented peptide, and that this
trimming process is stimulated by IFN-. Interestingly, these
proteasome-free cytosolic extracts did not trim the various
SIINFEKL-related peptides on their C termini to any significant degree.
In fact, N-terminal processing accounted for over 90% of the
metabolism of the 11-mer peptide in these extracts. Therefore, there
appears to be very little carboxypeptidase activity in these cells. It
is noteworthy that no carboxypeptidase has been described within the
cytosol of mammalian cells (41), although some such activity has been
reported in the ER (42, 43).
This lack of C-terminal processing provides biochemical support for our
prior conclusion that the proteasome generates the correct C terminus
of SIINFEKL and presumably also of most other presented peptides.
Accordingly, when the C terminus of the SIINFEKL was extended by 1-15
residues, its presentation was markedly inhibited by blockers of
proteasome function (29). This conclusion also is in accord with the
finding that IFN-, by altering the proteasome's peptidase
activities should enhance the production of peptides with hydrophobic
and basic C termini, exactly the types of peptides that bind
preferentially to the TAP transporter (15) and MHC class 1 molecules
(16). By contrast, when the N terminus of SIINFEKL was extended by up
to 25 residues, the cells could generate the antigenic peptide by a
non-proteasomal mechanism; and in HeLa or U937 extracts, we found that
the 11-residue precursor was shortened by progressive removal of single
residues, especially in IFN-
-treated cells. Several findings
indicate that LAP is the critical enzyme in this generation of the N
terminus of SIINFEKL. (i) The purified enzyme trimmed the 11-mer to
peptides of 8-10 residues in a very similar manner as the extracts of
HeLa cells treated with IFN-
. (ii) This release of the N-terminal
residues was inhibited by bestatin, an inhibitor of LAP, and the extent
of inhibition was similar with the extract and the pure enzyme. (iii)
This trimming is stimulated by IFN treatment; in fact, in the absence
of IFN-
, the antigenic peptide was not generated under the
conditions used here. (iv) Although three major aminopeptidase peaks
were detected in these extracts, only one, LAP, was induced by IFN-
,
and it alone was active in the processing of the 11-mer. This enzyme was identified by immunoblot analysis and sensitivity to bestatin. (v)
Moreover, the substrate preference of pure porcine LAP against 19 different amino acid-AMC substrates correlated well with that of the
IFN-induced enzyme in the human cell extracts. Thus, although IFN-
may induce other peptidases, LAP induction by itself seems to account
for all of the changes in peptidase activity and SIINFEKL trimming in
these extracts. These findings, however, do not exclude the possibility
that the non-induced aminopeptidases or endopeptidases may also be
involved in processing of other antigenic peptides or in the complete
digestion of most proteasomal products to amino acids.3
IFN- thus stimulates multiple steps in the MHC class I pathway,
which together should have additive or synergistic effects in promoting
antigen processing. In addition to inducing LAP, IFN-
enhances the
proteasome's capacity to generate peptides with the appropriate C
termini and induces the PA28 proteasome activator, which seems to favor
the generation of 8-10-mer peptides (28). By trimming longer
epitope-containing peptides, LAP should enhance the yield of peptides
of proper length for tight binding to MHC class I molecules. These
findings clearly suggest a collaboration between the proteasomes and
this cytosolic peptidase to enhance the efficiency of antigen
presentation. The coordinated induction of the TAP transporter and MHC
class I molecules by IFN-
should further increase the number of
presented peptides.
It appears likely that peptides released from the proteasome that are too long for MHC class I binding undergo N-terminal trimming in the cytosol by LAP prior to transport into the ER. The TAP complex is able to efficiently translocate peptides containing 5-16 residues (30). Once the peptides are transported, their N termini may be further trimmed in the ER (29, 32, 44), which contains aminopeptidases that can also process the N-terminal-extended SIINFEKL peptides. Alternatively, the peptides once in the ER, may be rapidly transported back into the cytosol by the ER retrograde-transport system (43, 45, 46). Unoccupied MHC class I molecules are associated with the TAP transporter, and if transported peptides are too long to bind tightly to the MHC molecule, they can be readily transported back into the cytoplasm for further trimming by LAP. Repeated aminopeptidase cycles should thus eventually generate peptides of appropriate length for association with MHC class I molecules. Once tight binding is achieved, it should prevent further transport back into the cytosol and further proteolysis.
These models apply if the antigenic peptides released by the proteasome are too large for optimal TAP transport or MHC class I binding. It remains uncertain to what extent 26 S proteasomes do in fact generate 8-mer or N-terminal-extended versions of SIINFEKL (or other antigenic peptides). Recently, Kisselev et al. (24)2 found that the 20 S and 26 S proteasomes degrade proteins to oligopeptides ranging from 3 to 25 amino acids long, in clear contrast to the proposal that proteasomes generate uniformly octapeptides according to a "molecular ruler" mechanism (27, 47). Less than 15% of the products were 8 residues long, while up to 15% are 10 amino acids or longer (24),2 and these peptides would clearly be potential substrates for processing by LAP for antigen presentation (if they have the appropriate C termini).
Such N-extended, longer versions of antigenic peptides have in fact
been detected in vivo. For example, efforts to purify the
transplantation antigen recognized by the alloreactive T-cell clone,
C-2, identified both the antigenic 8-mer and a 16-mer precursor (48,
49). Similarly, Uenaka et al. (50) found not only the 8-mer
peptide for the immunogenic BALB/c radiation-induced leukemia RLo1, but
also found a 10-mer. Purified proteasomes also were found to generate
N-terminal-extended peptides; e.g. among the peptides
released during the hydrolysis of ovalbumin (29) and of 22- and
41-residue fragments of ovalbumin (27) were the N-terminal-extended 9-mer and the 11-mer studied here (29). Although these reactions were
run under rather nonphysiological conditions, proteasomes clearly can
generate N-extended versions of antigenic peptides. The induction of
LAP by IFN- strongly suggests that the trimming of such peptides is
a rate-limiting step in the presentation of certain antigenic
peptides.
Implications for the Immunodominance of Antigenic
Peptides--
Antigenic proteins must contain a large number of
sequences that can potentially be presented on MHC class I molecules,
and it is unclear why the immune system mounts responses to a very limited number of these sequences (26). The present findings imply that
there are 4 or 5 proteolytic processes which influence whether or
not an antigenic peptide appears on the surface: (i) whether the
proteasome cleaves precisely at the correct C terminus of the epitope;
(ii) whether these particles release a peptide of sufficient length to
bind to MHC class I molecules; (iii) whether the proteasome by chance
also cleaves before the appropriate N terminus; if not, (iv) whether
the N-terminal residues flanking the epitope can be removed efficiently
by aminopeptidases. As shown in Table I, the presence of certain amino
acids in the N terminus of a peptide can make it a very good or very
poor substrate for cytosolic aminopeptidases. In fact, the N-terminal
residues differ up to 20-fold in their susceptibilities to
exopeptidases and to IFN-stimulated hydrolysis. Therefore, the presence
of residues which are released faster by LAP in the N-terminal-extended
region would lead to faster trimming, while residues which are poorly released should slow down its trimming to a size that binds to MHC
class I molecules; (v) whether cytosolic endopeptidases or exopeptidases destroy the correct epitope once formed.3 As
noted here, SIINFEKL is susceptible to cytosolic proteases, and it is
noteworthy that this degradation, unlike SIINFEKL generation, is not
stimulated by IFN-. Thus, this proteolytic step which may limit
antigen presentation seems to involve distinct protease(s) from those
involved in epitope generation.3 Identity of the enzyme(s)
that degrade antigenic peptides will be important to establish. It is
also noteworthy that we found no evidence for a peptide-binding
chaperone (40) that might bind and protect the mature peptide from
further digestion.
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FOOTNOTES |
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* This work was supported by research grants from the National Institute of General Medical Sciences and the Human Frontier Science Program (to A. L. G.) and a postdoctoral fellowship from the Deutsche Forschungsgemeinschaft (to J. B.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ To whom correspondence should be addressed. E-mail: agoldber{at}bcmp.med.harvard.edu.
2 A. F. Kisselev, T. Akopian, K. M. Woo, and A. L. Goldberg, submitted for publication.
3 J. Beninga, K. L. Rock, and A. L. Goldberg, submitted for publication.
1 The abbreviations used are: MHC, major histocompatibility complex; ER, endoplasmic reticulum; LAP, leucine aminopeptidase; IFN, interferon; HPLC, high performance liquid chromatography; AMC, aminomethylcoumarin; pkLAP, porcine kidney LAP.
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REFERENCES |
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