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*
Institute of Biochemistry, Medical Faculty, Charité, Humboldt University, Berlin, Germany; and
Division of Tumor Biology, Netherlands Cancer Institute, Amsterdam, The Netherlands
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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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1-7, 
1-7) (2, 3). The
catalytic sites are located within the two inner rings of the 20S
proteasome complex and are formed by three constitutive subunits,
1, 2, and 5 (i.e., delta, z, MB1). Importantly, the
active site bearing subunits can be replaced by the
IFN-
-inducible homologous subunits (i1/LMP2, i2/MECL1,
i5/LMP7), resulting in the formation of the, so-called,
immunoproteasome. Another IFN--inducible component of the proteasome
system is the proteasome modulator complex PA28 (4, 5, 6),
composed of the PA28 and PA28 subunits, which participates in the
generation of antigenic peptides required for MHC class I Ag
presentation. Previously, we have shown that expression of the PA28
subunit in mouse fibroblast B8 cells to a level similar to that
obtained as a result of IFN- induction resulted in the enhanced MHC
class I presentation of an influenza nucleoprotein and a murine CMV
(MCMV)4 immediate
early (IE) protein pp89-derived epitope (7). In agreement
with this and supporting the validity of in vitro digestion
experiments, we also showed that in vitro processing of a larger
synthetic polypeptide derived from the MCMV pp89 by 20S proteasome in
the presence or absence of PA28 is able to support the generation of
the pp89 MHC class I epitope with an efficiency comparable with that
observed in vivo (8). In
PA28-/- mice, lacking the PA28 and
PA28 subunits, CTL responses are impaired (9).
Peptides generated by the proteasome system in the cytoplasm are
translocated into the lumen of the endoplasmic reticulum (ER), where
they can associate with MHC class I molecules, a process that is
mediated by specific IFN--inducible peptide transporters named TAP.
TAP binds cytoplasmic peptides with a broad, but not indistinguishable
specificity, and translocates them into the ER in an ATP-dependent
fashion (10). In general, TAP prefers peptides of about 9
aa, similar to MHC class I molecules, but can also translocate smaller
and larger peptides albeit with lower efficiency (11, 12).
Up to now it is unclear which peptide fragments generated by the
proteasome ultimately enter the ER. Are these by definition peptides of
the correct size for MHC class I binding, or are these in some cases
precursors that require further trimming? In addition, the role of PA28
in this combined process is unresolved. Processing following
proteasomal cleavage could take place in the cytosol. Beninga et al.
(13) described a cytosolic IFN--inducible leucine
aminopeptidase that processed an epitope precursor in vitro. It remains
to be shown whether this or other cytosolic peptidases are involved in
Ag processing in vivo. In addition, peptide competition experiments for
TAP translocation suggest that N-terminal trimming may occur in the ER,
but no ER peptidase was isolated to date (14, 15).
In this study, we have followed the degradation of a MCMV pp89 fragment by proteasomes in the presence or absence of PA28. For the first time, peptide generation by the proteasome system and TAP translocation have been experimentally linked. We show that PA28 is inducing the formation of an 11-mer epitope precursor fragment that is arriving in the ER. In contrast, the generated 9-mer epitope was not detected in the ER. Consequently, the 11-mer precursor represents the immunorelevant peptide product that requires N-terminal trimming in the ER for MHC class I binding.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Mouse B8 fibroblast cells (16) were cultured in
IMDM supplemented with 10% FCS, 2 mM L-glutamine, and 10
U/100 µg/ml penicillin/streptomycin. Induction with 20 U/ml mouse
rIFN- (Boehringer Mannheim, Mannheim, Germany) was performed for
72 h. Human T3 cells (17) derived from TAP-deficient
T2 cells, transfected with rat TAP1 and TAP2a and
mouse EL4 cells were grown in HEPES-buffered RPMI 1640 (Biochrom,
Berlin, Germany) supplemented with 8% FCS. Human LCL 721 cells were
grown in RPMI 1640, 8% FCS (18).
Proteasome purification
The 20S proteasomes were isolated from IFN--induced (B8i) or
noninduced (B8) mouse B8 fibroblast cells. About 6 x
108 cells were harvested with
Ca2+/Mg2+-free medium and
homogenized in 5 vol buffer A (10 mM HEPES, pH 7.2, 80 mM KCl, 5 mM
MgCl2) plus 0.1% Triton X-100. The cleared
lysate was applicated onto DEAE Sephacel (Pharmacia, Freiburg,
Germany) and washed. Proteasomes were eluted with 500 mM KCl in buffer
A and concentrated by ammonium sulfate precipitation between 40 and
70% saturation. This protein fraction was further separated by
ultracentrifugation through 10–40% sucrose gradients (Beckman SW40
rotor, 15.49 h at 38,000 rpm; Beckman, Fullerton, CA). The
proteasome-containing fractions were pooled and applied to a ResourceQ
column (fast protein liquid chromatography; Pharmacia) and eluted with
a gradient of 100–500 mM KCl in 10 mM HEPES, pH 7.2, 5 mM
MgCl2. Rechromatography of isolated proteasomes
was performed by fast protein liquid chromatography on a MonoQ column
(Pharmacia).
Expression and purification of rPA28
Recombinant PA28 and PA28 were expressed in
Escherichia coli as GST-fusion proteins and purified, as
described previously (19).
Peptide digests and HPLC separation
For digestion of a synthetic 25-mer peptide modified from the
sequence of MCMV IE protein pp89, 5 µg peptide was dissolved in 100
µl HPLC buffer (20 mM HEPES, pH 7.8, 2 mM potassium acetate, 1 mM
dithioerythrit) and incubated with 0.5 µg proteasome for indicated
times at 37°C. Recombinant PA28 and PA28 were added in a
10-fold molar excess. Cleavage products were analyzed by reversed phase
(RP)-HPLC (System Gold; Beckman). Twenty microliters of digest were
applied on a Micra NPS ODS-I 1,5-µm column (Bischoff Chromotography,
Leonberg, Germany) and eluted with a flow rate of 1 ml/min and a
linear gradient of solution A (H2O, 0.1%
trifluoroacetic acid) and solution B (acetonitrile, 0.1%
trifluoroacetic acid). The gradient was performed at 30–100% solution
B in 15 min.
Peptide analysis by mass spectrometry (ms)
Samples were analyzed by RP-HPLC (HPLC system HP1100; Hewlett-Packard, Waldbronn, Germany) equipped with a µRPC C2/C18 SC 2.1/10 column (Pharmacia): eluent A, 0.05% trifluoroacetic acid; eluent B, 70% acetonitrile, 0.045% trifluoroacetic acid; gradient, 25%–63% eluent B in 30 min, 63–95% eluent B in 4 min; flow rate, 50 µl/min. Analysis was performed on line with an ion trap mass spectrometer (ThermoQuest, Egelsbach, Germany) equipped with an electrospray ion source. Each scan was acquired over the range m/z = 300-1300 in 3 s. The peptides were identified by their molecular masses calculated from the m/z peaks of the single or multiple charged ions and were confirmed by mass-spectrometric sequencing analyses.
Microsome isolation
Microsome isolation was performed as described (20).
Peptide iodination
Peptides (25 µg in 25 µl) were radiolabeled by Chloramine T-mediated iodination (10) with 1 mCi Na125I, and free iodine was separated on a 2-ml Dowex column. Iodinated peptides with a sp. act. of 30–40 µCi/µg were stored in PBS at -20°C.
Peptide translocation assays
Peptide translocation assays were performed essentially as described (10). Iodinated peptides (250 ng) were incubated with 5 µl T3 microsomes for 10 min at 37°C in the absence or presence of 10 mM ATP in 100 µl incubation buffer (5 mM HEPES, pH 7.3, 130 mM KCl, 10 mM NaCl, 1 mM CaCl2, 5 mM MgCl2, 2 mM EGTA). Translocation was stopped by adding 1 ml ice-cold incubation buffer, and untranslocated peptides were removed by pelleting the microsomes (5 min, 17,000 x g, 4°C) and subsequent washing with 1 ml incubation buffer. The pellet was lysed for 10 min on ice in 500 µl lysis buffer (150 mM Tris, pH 7.4, 150 mM NaCl, 5 mM MgCl2, 0.5% Nonidet P-40). The debris was removed by centrifugation (5 min, 17,000 x g, 4°C), and the supernatant was incubated with 25 µl Con A-Sepharose beads (Pharmacia) for 1 h at 4°C while rotating. The Con A-Sepharose was washed three times with washing buffer (150 mM Tris, pH 7.4, 500 mM NaCl, 5 mM MgCl2, 0.5% Nonidet P-40, 0.1% SDS), and the Con A-Sepharose-bound peptides were quantified by gamma counting.
Coupled peptide digest and translocation assay
Iodinated 25-mer peptide (4–6 µCi/translocation) was digested
with the appropriate amount of proteasome and with or without rPA28
(125–175 ng proteasome per µg peptide, PA28 and PA28 in a 10-
to 33-fold molar excess dependent on the sp. act.). Incubation buffer
was added to a total volume of 170 µl. Samples were incubated at
37°C, and 25-µl aliquots were taken at indicated time points.
Clasto-lactacystin -lactone (Boston Biochem., Boston,
MA) was added to 10 µM to prevent further degradation, and
2-µl aliquots were extracted with 30 µl phenol as digestion
references. The samples were splitted in two halves and directly added
to translocation assays with or without 10 mM ATP and 5 µl T3 (10
µl EL4, or 10 µl B8i) microsomes in 100 µl total volume.
Translocation assays were performed for 10 min at 37°C, as described
above. Con A-Sepharose-bound peptides were extracted with 0.2% SDS in
PBS by boiling for 5 min at 95°C and removed by centrifugation. The
supernatant was incubated with 1 µm endoglycosidase H (Boehringer
Mannheim) at 37°C overnight. Deglycosylated peptides were extracted
with 50 µl phenol, and 10-µl samples were separated by TLC on
Kieselgel 60 plates (Merck, Darmstad, Germany) using the following
separation system, n-butanol/pyridine/acetic
acid/H2O, 48.5:37.5:7.5:30, as described
(11). After separation, plates were dried and exposed to
Kodak X-AR5 films or to a phosphor imager (BAS Reader 2000, TINA 2.09
software; Fuji, Tokyo, Japan).
Analysis of translocated peptides by ms
A total of 50 µg 25-mer peptide was digested with 4 µg B8
proteasome for 4 h at 37°C. The sample was translocated into 450
µl EL4 microsomes, as described above. Glycosylated peptides were
bound to Con A-Sepharose and eluted with 250 mM
methyl-D-mannopyranoside in 150 mM Tris, pH 7.4, 500 mM
NaCl, 5 mM MgCl2. Peptides were deglycosylated
with 5 U N-glycosidase F (Boehringer Mannheim) at 37°C
overnight. Samples were filtered (Membrex 4 CA, 0, 2 µm; membraPure),
adjusted to 1% acetic actid, 10% acetonitrile, and bound to
C18 ziptips (Millipore, Eschborn, Germany).
Peptides were eluted with 70% methanol, 1% acetic acid, and directly
applied to the analysis by an ion trap mass spectrometer (LCQ;
ThermoQuest, Bremen, Germany) equipped with an electrospray ion source.
Ions with a mass/charge value of 662 ± 1 Da (corresponding to the
double-charged 11-mer peptide) were selected and fragmented by
collision-induced dissociation.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To combine proteasomal degradation with translocation by TAP, we
used a modified polypeptide originally derived from the MCMV IE protein
pp89 (Fig. 1
). An N-linked
glycosylation site was introduced in the presented epitope by changing
M11P12 to
N11A12 to facilitate
recovery of TAP-translocated peptides through the acquired
oligosaccharides. Two D-amino acids at the
extremities of the model peptide should prevent TAP-mediated
translocation of the input 25-mer (21). To confirm that
the modifications did not affect the degradation of the 25-mer model
peptide, the peptide was incubated with 20S murine proteasomes in the
absence or presence of rPA28/ for various time points. As shown
in Fig. 2, 20S proteasome is able to
degrade the model peptide and generates 11- and 9-mer fragments
containing the MHC class I epitope, as determined by ms. PA28 markedly
accelerates the formation of the 11- and 9-mer fragments within the
first hour of incubation. Note that the fragments are ultimately
destroyed upon long incubations (24 h) in the presence of PA28, whereas
free PA28 does not show any protease activity (Fig. 2H). A
comparison of degradation products generated from wild-type or modified
25-mer peptide (Table I) shows that the
modified 25-mer is a suitable substrate for the following studies. With
few exceptions, the main products and the products of interest
(9-mer/7–15 and 11-mer/5–15) were generated in both cases after 1 and
24 h.
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TAP translocates peptides from the cytoplasm into the ER lumen in
an ATP-dependent fashion. To test which of the substrates that include
the presented epitope are potential substrates for TAP-dependent
translocation, the 9-mer epitope, and the 11- and 25-mer input
fragments were radiolabeled with 125I at tyrosine
residues. The peptides were incubated with microsomes purified from
cells expressing rat TAP1/2a in the absence or presence of
ATP. In addition, microsomes from IFN--induced B8 cells (B8i) were
used in this assay. The translocated, and consequently glycosylated,
fraction was isolated by Con A-Sepharose, and the bound peptides were
quantitated (Fig. 3). Only the synthetic
11-mer peptide was efficiently translocated by TAP in the presence of
ATP. In contrast, no transport was observed for the 9-mer epitope and
the 25-mer input peptide. The same results were obtained with
microsomes from murine (EL4 cells) or human cells (LCL 721 cells) (data
not shown).
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Efficient generation of an epitope does not necessarily imply that
it is also efficiently appearing in the ER lumen and vice versa.
Therefore, we decided to combine the proteolytic digest with the
translocation system (Fig. 4). The
modified pp89 25-mer was incubated with immunoproteasome (B8i) and
rPA28/ for 1 h. Sequentially, translocation assays were
performed in the presence or absence of ATP. A simultaneous degradation
and translocation assay could not be used, since isolated microsomes
were not stable at 37°C for longer time periods even under isotonic
conditions (data not shown).
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, lane 2), which strongly suggests that, after proteasomal
degradation of the 25-mer, only the 11-mer is transported. A third band
in the second lane is unspecific since it is also found in
the assay without ATP (lane 1). Assays with synthetic
9- and 25-mer showed no signals over background level (data not
shown).
To proof the existence of the 11-mer in the ER, the 25-mer peptide was
degraded by B8 proteasome, and the generated peptides were translocated
into murine EL4 microsomes. Translocated peptides were isolated, as
described in Materials and Methods, deglycosylated by
N-glycosidase F, and analyzed by ms. The 11-mer precursor
could be detected in the ER lumen according to its mass, and the
identity was confirmed by ms/ms sequencing as shown in Fig. 5. Note that due to the
N-glycosidase F treatment, the asparagine residue involved
in the glycosidic linkage was changed into an aspartic acid. However,
no 9-mer or oxidized 11- mer could be detected by ms. These experiments
demonstrate that of the proteasomal processing products, only the
11-mer peptide is efficiently translocated.
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The 11-mer precursor peptide is more prominently generated in the
presence of PA28, as determined by ms (data not shown). To test whether
this peptide also enters the ER more efficiently, proteasomal
degradation was coupled with translocation assays. The pp89 25-mer was
incubated for up to 6 h with murine constitutive (B8) or
immunoproteasome (B8i) in the presence or absence of rPA28/. The
subsequent translocation into T3 microsomes was performed for 10 min in
the presence or absence of ATP. Samples of the degradation products and
the translocated peptides were separated by TLC.
The separation of the degradation products on TLC plates enabled us to
detect even small amounts of radiolabeled fragments (Fig. 6). The amount of the input peptide
decreased, and several degradation products appeared with ongoing
degradation. Surprisingly, not only the 11-mer, but also the 9-mer
could be detected after 30 min of incubation with both proteasome
subtypes B8 and B8i, respectively (Fig. 6A). It accumulated
as the incubation time increased. PA28 enhanced 20S proteasome
activity. In the presence of PA28, B8, and B8i proteasomes generated
higher amounts of 9- and 11-mer at the corresponding time points. Using
B8i proteasome plus PA28, the levels of 9- and 11-mer decreased during
extended incubation time (6 h), indicating further degradation of the
products. However, the amount of the input peptide decreased slower in
assays plus PA28. This result could be confirmed by ms, whereas it was
not observed with the wild-type substrate. The diminished total
turnover might be due to the D-amino acids at the
N and C terminus.
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). From the degradation samples taken
between 30 min and 6 h (Fig. 6), translocation products could be
determined in all translocation assays with ATP. They were separated
into two bands, with the lower one being the most prominent. No
translocation products were found in samples without ATP and at 0 min
of degradation. PA28 enhanced peptide generation, which resulted in
increased precursor translocation. After 6 h of incubation, the
11-mer was further degraded in the presence of PA28, and in consequence
less 11-mer product was translocated. Apart from the 11-mer, no other
translocated peptides could be detected. Due to our experimental
approach, the 11-mer epitope precursor is trapped in the ER by the
acquired carbohydrate chain. In vivo, however, it has to be trimmed by
an ER resident peptidase to allow efficient MHC class I binding.
Therefore, these experiments show that the immunodominant epitope of
the MCMV pp89 protein is generated as an N-terminally elongated
precursor by the proteasome, and that only this 11-mer peptide is
translocated by TAPs.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The proteasomal cleavage properties determine the peptide pool, which may be translocated by TAP. Recent data suggest that the majority of MHC class I-bound peptides is generated by the proteasome without need for additional processing (22). However, several MHC class I epitopes are generated by the proteasome as putative precursor peptides (14, 23).
For a steadily increasing number of epitopes, including pp89 (16, 24), it has been shown by us and others that, with regard to both quantitative as well as qualitative aspects, the in vitro processing experiments using purified 20S proteasoms reflect the in vivo situation with high fidelity (15, 16, 22, 25, 26). Using this experimental approach in combination with a direct translocation assay, this study shows that the MHC class I epitope of the MCMV pp89 protein is generated and translocated as a precursor peptide.
In our in vitro experiments using purified 20S proteasome and rPA28,
pp89 9- and 11-mer peptides could be detected after 30 min. Prolonged
incubation times such as 24 h resulted in the further degradation
of products generated at earlier time points (Fig. 2). This contrasts
with previous studies in which incubation times of several hours were
chosen to ensure total substrate turnover (24, 27). Given
that generated peptides were translocated forthwith into the ER and are
presented by MHC class I molecules within 1 h after virus
infection (28), only the products of a first round of
degradation would most likely fulfill in vivo conditions. It is
tempting to speculate that unknown factors might improve Ag
presentation by peptide channeling from proteasome to TAP. Further
investigations are on its way to address this challenging question.
The immunodominant 9-mer could hardly be detected in previous studies (24) or only in the presence of PA28 (8). As expected, the modified substrate shows a slightly different cleavage pattern than the wild-type 25-mer. In vivo and in vitro studies elucidated the influence of flanking residues on the usage of cleavage sites (29) (Kuckelkorn et al., unpublished results). The amino acid changes in the epitope sequence necessary for peptide recovery after translocation changed the nature of the products. Nevertheless, the peptides of interest were generated. In addition, the radioactive labeling of the substrate facilitated the detection of the generated epitope also in the absence of PA28. Constitutive (B8) and immunoproteasome (B8i) produced increased amounts of 9- and 11-mer in the presence of PA28. The effect of PA28 was even stronger for the immunoproteasome where these products decreased after 6 h.
Remarkably, PA28 does not change the cleavage pattern qualitatively, but accelerates the generation of distinct products. The same products were generated as without PA28, but at earlier time points. The epitope and its precursor were most efficiently generated by the immunoproteasome in the presence of PA28. These results were confirmed with the unmodified pp89 25-mer peptide and in studies with a p53 epitope (Kuckelkorn et al., unpublished results).
In case of the murine leukemia virus MCF8 epitope competition experiments recently showed that the epitope itself does not block the import of a reporter peptide, whereas the corresponding precursor peptides prevent translocation (13, 14). However, the ability of a peptide to inhibit the translocation of a reporter peptide does not necessarily imply that it is translocated itself. Consequently, only direct translocation assays are suited to determine whether a peptide is translocated or not.
In a first approach, we tested the translocation efficiencies of the pp89 epitope, its putative 11-mer precursor, and the 25-mer substrate peptide in in vitro translocation assays. Only the 11-mer was actively translocated according to the length and sequence specificity of TAP. The sequence specificity with regard to the C-terminal residue of the peptide differs in various species. Mouse TAP and rat TAP1/2a prefer peptides with hydrophobic or aromatic C-terminal residues, whereas human TAP and rat TAP1/2a also translocate peptides with basic C termini (30 , reviewed in Ref. 31). With a hydrophobic amino acid (leucine) at the C terminus, the 11- mer was translocated by murine, rat, and human TAP. However, the 9-mer was not translocated due to proline at position 2. A proline residue at the first three positions of the peptide negatively influences transport (32, 33). In the putative precursor, this residue is situated at position 4, and therefore without any influence on ER translocation. It is noteworthy that proline at position 2 and leucine at position 9 of the 9-mer correspond to the MHC class I allele H-2Ld-specific anchor residues (34, 35, 36). In addition, phenylalanine at position 4 is the main TCR contact residue (37). The 25-mer was not translocated due to the D-amino acids at the extremities, which prevent peptide translocation (21). Thus, to trigger a MHC class I-dependent CTL response, different requirements have to be conducted by the peptide at the level of TAP-dependent translocation and MHC class I binding.
To answer the question as to which degradation products are translocated by TAP, we combined for the first time proteasomal degradation with a TAP-dependent translocation system. A more detailed analysis of the translocated peptides by TLC identified the translocated degradation product as the 11-mer. The translocation products from synthetic 11-mer and the pp89 peptides generated by the proteasome separated identically on TLC plates. Most importantly, the nature of the translocated 11-mer was proven by ms. The results are in perfect agreement with the translocation efficiencies of the synthetic epitope and precursor.
The fact that translocated pp89 11-mer precursor accumulates in the ER and is not further trimmed in our approach (even in microsomes derived from B8i cells expressing the correspondent MHC class I haplotype) could be explained in that the carbohydrate chain impairs MHC class I binding and protects the peptide against further trimming.
Translocation of peptides degraded up to 6 h revealed that the
precursor is translocated at the same extent as it is generated in
vitro. Even at longer incubation times, no 9-mer was translocated. A
combination of three IFN--inducible components involved in Ag
presentation, immunoproteasome, PA28, and TAP promotes the best peptide
supply into the ER.
The pp89 epitope is translocated into the ER as a precursor, whereas it is presented at the cell surface by MHC class I molecules as a 9-mer. This implies an additional processing step in the ER lumen. It is formally possible that a few 9-mer peptides enter the ER, despite the steric hindrance of a proline residue at position 2. However, there is no evidence that those would play a role in Ld presentation compared with a pathway that comprises very efficient translocation of a precursor peptide, followed by ER trimming. Nevertheless, cytosolic trimming of precursors may not be excluded for other peptides not carrying a proline residue at position 2.
The ER peptidase involved in MHC class I ligand trimming could not be identified until now, but has been predicted for some time. The variability of the N termini of peptides eluted from MHC class I molecules led to a model in which the N terminus may be processed while the C terminus is fixed by the MHC class I molecule as an anchor residue (38, 39). Experiments with signal sequence-linked peptides gave first experimental hints for ER trimming (40, 41). These peptides were imported independently of TAP into the ER, and C-terminal epitopes were liberated. However, an imprecise cleavage by the signal peptidase could not be excluded.
Recently, Paz et al. (42) detected indirectly proteolytic intermediates in the ER. The Kb-restricted peptide from OVA was expressed at the C terminus of a type II ER protein. Precursor peptides could be detected in the cytosolic and ER fractions, whereas the 8-mer epitope was only found in the ER in the presence of the corresponding MHC class I molecules. In the absence of the MHC class I molecules, a 9-mer could be detected as a proteolytic intermediate in the ER. These data suggest that peptide trimming may take place in the peptide-loading complex or in an MHC class I-bound stage.
We now show in a proteasome degradation system coupled to TAP translocation that a MHC class I peptide extended N-terminally by 2 aa is selected for ER import by TAP, whereas the correct sized fragment is excluded. These data illustrate the importance of a coupled degradation/translocation system.
An ER resident amino peptidase as a further component of the Ag-processing machinery would mediate between the selectivity of TAP and the length and sequence specificity of the MHC class I molecule.
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Acknowledgments |
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Footnotes |
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2 Current address: Biomolecular Chemistry, ZMBH, Ruprecht-Karls University Heidelberg, 69120 Heidelberg, Germany.
3 Address correspondence and reprint requests to Dr. Peter-M. Kloetzel, Institute of Biochemistry, Medical Faculty, Charité, Humboldt University, Monbijoustrasse 2a, 10117 Berlin, Germany. E-mail address: p-m.kloetzel{at}charite.de
4 Abbreviations used in this paper: MCMV, murine CMV; ER, endoplasmic reticulum; IE, immediate early; ms, mass spectrometry; RP, reversed phase.
Received for publication May 1, 2000. Accepted for publication June 1, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-interferon-inducible activator of the multicatalytic protease. J. Biol. Chem. 269:20727. in antigen presentation. Nature 381:166.[Medline]
can stimulate post-proteasomal trimming of the N-terminus of an antigenic peptide by inducing leucine aminopeptidase. J. Biol. Chem. 273:18734. stimulation modulates the proteolytic activity and cleavage site preference of 20S mouse proteasomes. J. Exp. Med. 179:901.. Eur. J. Immunol. 25:2605.[Medline]
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Y. Samino, D. Lopez, S. Guil, P. de Leon, and M. Del Val An Endogenous HIV Envelope-derived Peptide without the Terminal NH3+ Group Anchor Is Physiologically Presented by Major Histocompatibility Complex Class I Molecules J. Biol. Chem., January 9, 2004; 279(2): 1151 - 1160. [Abstract] [Full Text] [PDF]
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W. M. Cohen, A. Bianco, F. Connan, L. Camoin, M. Dalod, G. Lauvau, E. Ferries, B. Culmann-Penciolelli, P. M. van Endert, J. P. Briand, et al. Study of Antigen-Processing Steps Reveals Preferences Explaining Differential Biological Outcomes of Two HLA-A2-Restricted Immunodominant Epitopes from Human Immunodeficiency Virus Type 1 J. Virol., September 11, 2002; 76(20): 10219 - 10225. [Abstract] [Full Text] [PDF]
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R. Holtappels, D. Thomas, J. Podlech, and M. J. Reddehase Two Antigenic Peptides from Genes m123 and m164 of Murine Cytomegalovirus Quantitatively Dominate CD8 T-Cell Memory in the H-2d Haplotype J. Virol., January 1, 2002; 76(1): 151 - 164. [Abstract] [Full Text] [PDF]
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D. Lopez, Y. Samino, U. H. Koszinowski, and M. Del Val HIV Envelope Protein Inhibits MHC Class I Presentation of a Cytomegalovirus Protective Epitope J. Immunol., October 15, 2001; 167(8): 4238 - 4244. [Abstract] [Full Text] [PDF]
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) by
M11-N11 and P12-A12
exchanges. Amino acids 1 and 24 of the 25-mer were substituted by the
corresponding D-amino acids.
) or minus (
) ATP. T3
data are represented by filled boxes; B8i data by hatched boxes.
The data represent the mean ± SD of three independent
experiments.
