By
From the * Zentrum für Experimentelle Medizin (ZEM), Institut für Biochemie, Charité, Humboldt
Universität zu Berlin, 10117 Berlin, Germany; and the Max von Pettenkofer Institut, 80336 München, Germany
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Abstract |
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Proteasomes generate peptides bound by major histocompatibility complex (MHC) class I
molecules. Avoiding proteasome inhibitors, which in most cases do not distinguish between
individual active sites within the cell, we used a molecular genetic approach that allowed for
the first time the in vivo analysis of defined proteasomal active sites with regard to their significance for antigen processing. Functional elimination of the /low molecular weight protein
(LMP) 2 sites by substitution with a mutated inactive LMP2 T1A subunit results in reduced cell
surface expression of the MHC class I H-2Ld and H-2Dd molecules. Surface levels of H-2Ld and
H-2Dd molecules were restored by external loading with peptides. However, as a result of the
active site mutation, MHC class I presentation of a 9-mer peptide derived from a protein of
murine cytomegalovirus was enhanced about three- to fivefold. Our experiments provide evidence that the
/LMP2 active site elimination limits the processing and presentation of several
peptides, but may be, nonetheless, beneficial for the generation and presentation of others.
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Introduction |
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Using proteasome-specific inhibitors, the proteasome
system has been shown to be involved in antigen processing and to represent the major source for the generation
of MHC class I peptides (1). The 20S proteasome is an
NH2-terminal nucleophile hydrolase possessing an active
site threonine residue (5). It is a cylinder-shaped particle
composed of four stacked rings of seven subunits each. In
eukaryotes, the seven different type subunits occupy positions in the two outer rings, whereas the two inner rings
are formed by seven different
type subunits (6). The proteolytic activity is restricted to the lumen of the cylinder and
is mediated by three of the seven
type subunits, i.e., subunits
(
1), MB1 (
5), and Z (
2) (parentheses, new nomenclature according to Groll et al., reference 7). Therefore, in
total, the 20S proteasome complex possesses six active sites
within the two inner
rings. By induction with the cytokine IFN-
, the active site bearing constitutive
subunits
are replaced by their IFN-
-inducible counterparts low
molecular weight protein 2 (LMP2)1 (i
1), LMP7 (i
5),
and MECL-1 (i
2) during proteasome assembly (3, 8, 9).
Of these, LMP2 (i
1) and LMP7 (i
5) are encoded within
the MHC class II region in the direct neighborhood of the TAP1 and TAP2 peptide transporter genes (10, 11).
MECL-1 (i
2) is encoded outside the MHC locus, but its
incorporation into the 20S proteasome complex is guaranteed through the presence of LMP2 (i
1; reference 12).
The IFN-
-induced replacement of subunit
(
1) by
LMP2 (i
1), subunit MB1 (
5) by LMP7 (i
5), and Z (
2) by MECL-1 (i
2) results in changes of the hydrolytic activities as monitored with short fluorogenic peptide substrates (13, 14). In addition, the incorporation of these subunits strongly alters the cleavage site preferences of the 20S
proteasome in vitro (14, 15). As a consequence, a different
set of peptides products is generated by the 20S proteasome. Under physiological conditions, the ratio between
constitutive and cytokine-modified proteasomes complexes
changes only slowly. Accordingly, the abundance of certain
peptide products as well as their quality will gradually change during the time course of IFN-
induction. Indeed,
targeted deletion of LMP2 (i
1) and LMP7 (i
5) in mice
caused alterations in antigen presentation, emphasizing the
importance of these subunits for the generation of at least
certain MHC class I antigens (16, 17). Using proteasome
inhibitors, it has been shown that the inhibition of some of
the proteasomal peptidase activities affects the processing of
MHC class I antigens. (1, 18). However, there exists little
active site specificity of the available proteasome aldehyde
inhibitors. Even the active site specificity of lactacystin demonstrated in vitro is difficult to control in cell experiments since, depending on the experimental condition, lactacystin affects more than one type of active site (19, 20).
Therefore, experimental setups using proteasome inhibitors
in most cases do not allow one to draw any conclusions on
the functional importance of a specific active site for the
generation of a defined MHC class I antigen. Such knowledge is, however, important to better understand the basic
rules of antigen processing and to develop strategies that
may allow either up- or downregulation of the generation of a defined antigenic peptide.
To overcome these problems, we made use of a recently
described mutation in the nonconstitutive LMP2 (i1) subunit in which the NH2-terminal active site threonine was
replaced by alanine (21). This T1A mutation resulted in the
impairment of correct maturation by autocatalytic processing of the subunit and rendered an proteolytically inactive
LMP2 subunit. In this study, we used the inactive mutant
to study the functional importance of the
/LMP2 (
1/
i
1) active sites with regard to MHC class I antigen presentation. Overexpression of the mutant LMP2 T1A subunit
in mouse fibroblast cells resulted in an effective replacement of the proteolytically active
(
1) subunit. As a consequence, the mutant LMP2 T1A cells contain proteasomes
in which two of the six active sites of the 20S proteasome
complex are eliminated. Our experiments demonstrate that
the deletion of these active sites generally limits the production of peptides bound to H-2Ld and H-2Dd molecules.
At the same time, the mutation enhances the generation and presentation of an H-2Ld epitope derived from the cytomegalovirus pp89 protein.
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Materials and Methods |
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Cell Lines.
The BALB/c-derived mouse fibroblast cell lines C4 and B8 were used. The B8 cell line, which is derived from the C4 cells, constitutively expresses the IE I pp89 of the murine cytomegalovirus (22). The B8 cell line was subcloned by limiting dilution and one clone was chosen as recipient for the transfection experiments. The generation of the cDNA constructs of LMP2 and LMP2 T1A, transfection by conventional calcium phosphate precipitation and selection are described in detail in reference 21.Purification of 20S Proteasomes and Assay of Proteolytic Activity.
20S proteasomes were purified using standard procedures (21). Vmax and Km values were determined using the fluorogenic peptide substrates Bz-Val-Gly-Arg-7 Amido-4-methylcoumarin (MCA), Z-Gly-Gly-Leu-MAC, Suc-Leu-Leu-Val-Tyr-MCA, and Methoxysuccinyl-Gly-Leu-Phe-MCA (Bachem, Heidelberg, Germany). The peptides were used in concentrations from 5 to 200 µM and incubated with 1 µg/ml proteasome in 200 µl of 50 mM Tris-HCl, pH 7.5, 25 mM KCl, 10 mM NaCl, and 0.1 mM EDTA (assay buffer) for 1 h at 37°C as described before (14). Fluorescence intensity was measured at an excitation wave length of 390 nm and an emission wave length of 460 nm in a SLT Fluostar spectrofluorometer. Data were analyzed according to Lineweaver and Burk. All assays were performed in triplicate and repeated three times.Western Blotting.
15 ng of purified proteasomes were separated by SDS-PAGE and blotted as described (21). The blots were incubated with either LMP2- orNorthern Analysis.
The poly A+ mRNA of the cell lines C4, B8, B8-LMP2, control, and B8-LMP2 T1A was prepared using commercially available kits (Quiagen, Darmstadt, Germany). 3 µg of mRNA was applied to each lane. Agarose gel electrophoresis, blotting, labeling of the cDNA fragments, and hybridization was performed according to standard procedures (23). We used three different cDNA fragments simultaneously, a PstI/HindIII fragment (bp 297-815), a HindIII/PstI fragment (bp 815-1,314), and a PstI to 3' fragment (bp 1,314-1,788). After hybridization and washing the blot was quantitated and visualized with a PhosphorImager (Molecular Dynamics, Krefeld, Germany).Flow Cytometry.
Cells were removed from culture dishes with calcium- and magnesium-free medium, washed, and stained according to standard protocols with the monoclonal antibodies 19/191 (anti-H-2Dd), 3-25.4 (anti-H-2Dd; PharMingen, San Diego, CA), 28-14-8S (anti-H-2Ld), 28-14-8 (anti-H-2Ld; PharMingen), and 15-5-5S (anti-H-2Kd), and a sheep anti-mouse F(ab)2-FITC conjugate as a second-stage reagent. The analysis was performed with a FACSCAN® flow cytometer and LYSIS IITM software (Becton Dickinson, Heidelberg, Germany).Acid Elution of Natural Peptides and MHC Class I Peptide Binding Assay.
For peptide extraction and external-loading peptides we followed the procedure as described previously (24, 25). B8 cells (2 × 109) were separated from the culture dishes with calcium- and magnesium-free medium and washed with PBS to remove serum proteins. The cells were incubated for 15 min on ice in 0.1% TFA/H2O, sonicated, and kept on ice for another 15 min. Cells were centrifuged for 15 min at 15,000 rpm and the supernatant was collected. High molecular mass material was removed by centrifugation at 4°C through a 10-kD Centricon filter (Amicon Corp., Easton, TX). The peptides were concentrated by Speed Vac centrifugation to a final concentration of ~5 mg/ml as judged by OD 280. B8 cells and the transfectants were cultured for 18 h at 27°C in the presence of either 25 µg/ml of the synthetic peptide YPHFMPTNL (H-2Ld epitope of pp89) or ~25 µg/ml of peptides extracted from B8 cells by acidic elution. Cells were removed from culture dishes with calcium- and magnesium-free medium and incubated at a density of 106 /ml for 1 h on ice in PBS containing the peptides in concentrations as described above. After 1 h, cells were resuspended in serum-free medium containing the peptides and left for 2 h at 37°C at a density of 2 × 105 /ml. Staining of the surface level of MHC class I molecules was performed as described above.Cytolytic Assays.
Target cells for cytolytic assays were labeled for 90 min with Na251CrO4. A standard 4-h cytolytic assay was performed in triplicate with 1,000 target cells and the indicated numbers of effector cells in two- or fourfold dilution steps as detailed in reference 26. All experiments were performed three times in triplicate cultures with two clones of each transfectant, except for the LMP2 T1A transfectant where four clones were analyzed. ![]() |
Results and Discussion |
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To investigate the consequence of a defined active site elimination in the mouse 20S proteasome
complex, the murine fibroblast cell line B8 was stably
transfected with cDNAs either encoding a wild-type LMP2
or a mutated LMP2 subunit in which the active site threonine 1 residue was exchanged against alanine by site-directed mutagenesis (21). Overexpression of the LMP2 or LMP2
T1A subunits results in efficient incorporation of these subunits into the 20S proteasome complex (Fig. 1). The incorporation of the LMP2 proteins is associated with an almost
complete exchange against subunit . Accordingly, by immunoblotting with anti-
antibody only after overexposure
of the enhanced chemiluminescence blot, negligible amounts
of residual
subunit could be identified in 20S proteasomes of
B8-LMP2 and B8-LMP2 T1A cells. The slower electrophoretic mobility of LMP2 T1A also demonstrates that the
NH2-terminal prosequence is only partially cleaved, resulting
in an NH2-terminal extension of the subunit. Thus, overexpression of both the functional LMP2 subunit and the
LMP2 T1A active site mutant subunit and the concomitant elimination of the active site bearing
subunit from the
20S proteasome complexes allows production of a B8 cell
line whose proteasome population possesses only four, instead of six, active sites. Interestingly, the functional elimination of this active site had no obvious phenotypic effect
on the B8 fibroblast cells and appeared not to affect their
growth rate.
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The effect of the active site mutation
on the proteolytic activities of the 20S proteasome was
tested by analyzing the peptide-hydrolyzing activities of
20S proteasomes from B8, B8-LMP2, and B8-LMP2 T1A with short fluorogenic peptide substrates (Table 1). Independent of the substrates used, neither the substitution of
subunit by LMP2 nor the elimination of this active site
by incorporation of LMP2 T1A has a significant effect on
the Vmax of the 20S proteasome. This holds true for the
trypsin-like activity monitored with the substrate Bz-Val-Gly-Arg-MCA as well as for the chymotrypsin-like activity
measured with Z-Gly-Leu-Leu-MCA and Suc-Leu-Leu-Val-Tyr-MCA. Only in using MeOsuc-Gly-Leu-Phe-MCA was a reduction in Vmax by a factor of 2.9 measured
in the B8-LMP2 T1A mutant. Also, the Km value, the
measure for the binding affinity of substrates, was only
moderately influenced by subunit substitution or active site
mutation. For all substrates, we monitored an approximately twofold increase in the Km for the LMP2 T1A proteasome. One possible reason for the observed increase in
Km values in the LMP2 T1A mutant could be that the
NH2-terminal extension of 8-10 amino acids of the mutant
subunit influences the accessibility of the other active sites
and hence the substrate binding affinity. Apart from this,
the data suggest that the active site under investigation has
little effect upon the trypsin and chymotrypsin-like peptide substrates, which is in agreement with the previous finding
that the
/LMP2 site affects the peptidyl glutamyl peptide-hydrolyzing activity (PDGH activity) of the proteasome
complex. This activity is completely eliminated in these
cells (data not shown). On the other hand, these data show
that the different hydrolyzing activities, as monitored with
unphysiologically short substrates, are in fact overlapping
and that the attractive model of three different proteolytic specificities each mediated by one of three pairs of active
sites is perhaps too simple. In a recent investigation, Eleuteri and coworkers (27) came to a similar conclusion by
showing that short peptide hydrolyzing activities are overlapping and that different active sites cleave more than one
type of short fluorogenic substrate. Interestingly, the incorporation of the LMP2 subunit into 20S proteasomes as such
and not its activity seems to affect the enzymatic characteristics of the active sites of the neighboring subunit Z (
2)
and the more distant subunit MB1(
5). This suggests once
more (14) that the incorporation of this IFN-
-inducible subunit may also influence the structure function relationship within the proteasome complex.
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To investigate the effect of the /LMP2
active site mutation on the generation of antigenic peptides
in vivo, we analyzed the cell surface expression of MHC
class I molecules whose assembly and efficient transport to
the cell surface is dependent on the loading with suitable
peptides. Flow cytometric analysis of several independent
B8-LMP2 T1A cell clones with allele-specific antibodies revealed an ~40% reduction in the cell surface expression
of the H-2Dd and H-2Ld molecules when compared with
B8, B-LMP2, or B8 mock-transfected control cells (Fig. 2).
No difference in cell surface expression was found for the
H-2Kd molecules (data not shown). That this is not a clonal
effect is demonstrated by the finding that identical data
were obtained with different B8-LMP2 T1A cell lines.
These results suggest that the elimination of the two active
sites restricts the overall quality of peptide generated, thus
possibly limiting the supply of peptide and, in consequence, negatively affecting MHC class I molecule assembly and cell surface expression.
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To test this hypothesis we took advantage of the observation that MHC class I molecules can reach the cell's surface without prior peptide binding when cells are incubated at 27°C and that empty MHC class I molecules can
be stabilized by binding of externally added peptides (24,
25). B8-LMP2 T1A cells were therefore loaded either with
a synthetic 9-mer peptide that binds to H-2Ld or peptides
extracted from B8 cells. As expected from its binding characteristics, the synthetic 9-mer peptide restores the level of
H-2Ld, but not that of H-2Dd on B8-LMP2 T1A cells (Fig.
3 C). Furthermore, peptides extracted from nontransfected
B8 cells were able to stabilize the levels of both MHC alleles on the surface of B8-LMP2 T1A cells (Fig. 3 D).
These data demonstrate that it is indeed the lack of peptides that is responsible for reduced MHC class I expression on
the surface of B8-LMP2 T1A cells. In support of this, pulse
chase experiments and immunoprecipitation of H-2Dd and
H-2Ld molecules showed that these molecules are equally
well expressed in all cell lines analyzed (data not shown). It
may be argued that reduced temperatures can increase
MHC expression independent of peptide supply. However, under the same experimental conditions, the number
of Kd molecules does not increase at the cell surface at
27°C, even when peptides extracted from B8 cells are externally loaded.The elimination of active sites in the 20S
proteasome complex therefore decreases the general
amount of peptides available for binding to MHC class I
H-2Dd and H-2Ld molecules. In contrast, the level of H-2Kd
molecules is not reduced. Interestingly, all three haplotypes possess similar preferences for the COOH-terminal anchor
residue but differ with regard to the residue preference at
position 2 of the epitope. This indicates that the functional
importance of the activity of the /LMP2 varies depending
on the type of peptide products that have to be generated
for binding to a given MHC class I haplotype. In addition,
despite the fact that the peptide hydrolyzing activities of
the different active sites of the 20S proteasome are overlapping as deduced from in vitro data obtained with short fluorogenic peptide substrates (27), the
/LMP2 active sites
exert a specific cleavage property that is responsible for the
in vivo generation of a specific peptide quality from natural
protein substrates.
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So far the experiments showed that the functional elimination of two defined active sites in the 20S proteasome complex affects the quality of peptide generation and results in the downregulation of certain, but not all, MHC class I molecules. To determine how far the active site mutation influences the presentation of a specific peptide, we analyzed the different transfectant B8 cell lines with regard to their ability to present an immunodominant 9-mer peptide of the murine CMV (MCMV) pp89 to a H-2Ld-specific T cell line in a cytotoxicity assay. As shown in Fig. 4, B8 cells and B8 control-transfectant cells were lysed to the same extent, whereas B8-LMP2 cells were slightly less susceptible to lysis. In contrast, three- to fivefold less pp89/H-2Ld-specific cytotoxic T cells were required to lyse the B8-LMP2 T1A cells.
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To exclude the possibility that increased pp89 expression
in B8-LMP2 T1A cells was responsible for the observed effect, we investigated the expression of pp89 by Northern
blot analysis since pp89 is quite stable at the posttranslational level (22). As shown in Fig. 4 B, no significant differences in the expression of pp89 can be detected in the investigated cell lines. Thus, despite the elimination of two
active sites, sufficient pp89 antigen is generated to allow an
increase in peptide-specific MHC class I presentation. Although in vitro experiments do not necessarily reflect the
in vivo situation, it is interesting to note that in vitro digestions experiments of the pp89 25-mer synthetic polypeptide harboring the 9-mer epitope (15) show that the improved MHC class I presentation may be due to altered
proteasomal processing properties. Although /LMP2 proteasomes have the tendency to destroy the epitope, mutant
LMP2 T1A proteasomes do not use the internal cleavage
site and thus seem to preserve the epitope (Ruppert, T.,
unpublished observations). In consequence, the increased
maximum of lysis observed may be due to an increase in
specific peptide supply. Considering that the overall H-2Ld
surface expression is reduced in B8-LMP2 T1A cells, these
experiments represent the first example that the specific
elimination of a proteasomal active site, in this case
/LMP2,
may be beneficial for presentation of certain class I epitopes,
despite reduced MHC class I expression.
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Footnotes |
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Address correspondence to P.-M. Kloetzel, Zentrum für Experimentelle Medizin, Institut für Biochemie- Charité, Humboldt Universität zu Berlin, Monbijoustrabe 2, 10117 Berlin, Germany. Phone: 030-2802-6382; Fax: 030-2802-6608; E-mail: kloetzel{at}rz.charite.hu-berlin.de
Received for publication 2 October 1997 and in revised form 19 December 1997.
The present address of Marcus Groettrup is Kantonsspital St. Gallen, Laborforschungsabteilung, 9007 St. Gallen, Switzerland.This work was supported in part by the Deutsche Forschungsgemeinschaft grant Kl 427 9-2 and by the European Community grant BIO 4-CT97-0505 to P.M. Kloetzel and U.H. Koszinowski.
Abbreviations used in this paper LMP, low molecular weight protein; MCA, amido-4-methylcoumarin; MCMV, murine CMV.
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