(Received for publication, June 2, 1995; and in revised form, July 27, 1995)
From the
Antigenic peptides presented on major histocompatibility complex
(MHC) class I molecules to cytotoxic T cells are generated in the
cytosol by the 20 S proteasome. Upon stimulation of antigen presenting
cells with interferon-, two constitutive subunits of the 20 S
proteasome are replaced by the MHC-encoded subunits low molecular mass
polypeptide (LMP) 2 and LMP 7. In addition the expression of the two
subunits of the 11 S regulator of the 20 S proteasome (PA28) are
increased. As the function of LMP2 and LMP7 in antigen presentation is
still controversial, we tested whether these subunits might operate by
modifying proteasome activation through the 11 S regulator. We strongly
overexpressed the two LMP subunits separately or together by
transfection in murine fibroblasts. Isolated 20 S proteasomes from LMP
transfectants were applied in digests of a 25-mer peptide in the
presence or absence of a purified preparation of 11 S regulator from
rabbit erythrocytes. Analysis of the cleavage products by high
performance liquid chromatography and electrospray mass spectroscopy
revealed marked differences in the peptide product profile in
dependence on the LMP2 and LMP7 content. While the 11 S regulator did
not preferentially activate LMP2 or 7 containing proteasomes, the
binding of the 11 S regulator to any of the proteasome preparations
markedly changed both the quality and quantity of peptides produced.
These results suggest that the 11 S regulator increases the spectrum of
peptides which can be generated in antigen presenting cells.
In the course of a viral infection, the production of antigenic
peptides from intracellular viral proteins has to meet high demands: in
order to fit into the groove of major histocompatibility complex (MHC) ()class I molecules these peptides need to have a defined
length of 8 or 9 residues including fixed amino acids as anchor
residues. For the analyzed murine and human MHC haplotypes the C
terminus is either an aliphatic (Leu, Val, Ile), aromatic (Tyr), or
basic (Arg, Lys) residue(1, 2) . A further fixed
consensus amino acid lies in position 2 or 5, and even residues at
nonanchor sites are not arbitrary(3) . The viral peptide has to
meet the less stringent selectivity of TAP transporters(4) ,
and it cannot cross-react with a self-peptide for T cell receptor
binding as the T cells of that specificity are eliminated during
negative selection in the thymus(5) .
There is increasing
evidence that the 20 S proteasome, also called multicatalytic
proteinase, is responsible for generating antigenic peptides. The 20 S
proteasome is the major cytosolic endoprotease in
eukaryotes(6, 7, 8) . These 700-kDa protease
complexes, which constitute 0.5-1% of total cell protein, consist
of 14 different subunits ranging in molecular mass from 21 to 32 kDa
and with isoelectric points from 3 to 10, as evidenced by
two-dimensional analysis on NEPHGE-PAGE gels(9) . The subunits
can be classified as and
type based on their homology to
the two different subunits,
and
, of an ancestral proteasome
found in the archaebacterium Thermoplasma
acidophilum(10) . Seven
and seven
subunits
each form two rings stacked in the order
-
-
-
to
build the cylinder-shaped complex. Among the
type subunits, LMP2
and LMP7, which are encoded in the vicinity of the peptide transporter
genes in the MHC II complex (11, 12, 13, 14) , are induced by
the stimulation of cells with interferon-
, and they replace their
constitutive counterparts, designated delta and MB-1, in the
complex(15, 16, 17, 18) .
Two
further genes which are up-regulated by IFN- are not part of the
20 S proteasome itself but encode the two subunits constituting the
``11 S regulator'' (REG) or ``PA 28'' which is a
potent activator of the 20 S
proteasome(19, 20, 21, 22, 23, 24) .
Freshly isolated 20 S proteasomes are ``latent'' when assayed
with tri- or tetrameric standard fluorogenic peptide substrates.
Depending on the N-terminal amino acid from which a fluorescent leaving
group like MCA is cleaved by the proteasome, the REG activates the
proteasome 20-50-fold (Suc-LLVY-MCA, (Z)-LLE-
NA),
10-fold (PFR-MCA), or 3-fold (GGF-MCA). The REG has been isolated from
human blood as a hexa- or heptameric 180-kDa particle consisting of two
subunits with apparent molecular weights of 29 and 31 in SDS-PAGE. In
evolution both subunits are higly conserved with about 90% amino acid
sequence identity between human and rat. The 29- and 31-kDa subunits
may be functionally different as they display only about 50% identity
between each other(24, 25) . Electron microscopy has
shown that the ringshaped REG binds to the
-end plates of the
proteasome(26) . It thus competes with another complex
activator, called the 19 S regulator, for binding to the 20 S
proteasomes(27) . However, in contrast to the reversible
association between REG and 20 S proteasome which is energy
independent, formation of the 26 S proteasome out of the 20 S
proteasome and the 19 S regulator is ATP-dependent(28) . At
least four of the 13-15 subunits of the 19 S regulator belong to
a novel family of ATPases, and one subunit has been shown to be the
receptor of ubiquitin, crucial for the function of the 26 S protease in
degrading ubiquitinated proteins(29) .
Interferon-
potentiates antigen presentation on MHC class I molecules (30) by increased transcription of MHC class I and TAP genes.
The finding that two subunits of the proteasome and of the REG are
IFN-
inducible is a strong indication that the proteasome is
involved in the production of antigenic peptides. In fact, proteasome
inhibitors prevent an in vivo production of peptide ligands
for MHC class I molecules from ovalbumin(31) . Proteins or
peptides cleaved in vitro by the 20 S proteasome have been
found to yield antigenic peptides(32, 33) . Mice
deficient for the LMP7 gene show a decrease in MHC class I surface
expression on lymphocytes and the in vitro stimulation of
HY-antigen-specific T cells was reduced(34) . LMP2-deficient
mice, in contrast, are not reduced in MHC class I expression but show a
diminution of CD4
8
T cells. Upon
infection with influenza A virus, these mice show a reduction in the
frequency of precursors of antigen-specific cytotoxic T cells while no
change in cell number was noted in Sendai virus infection(35) .
It appears that the presence of LMP2 and LMP7 subunits in the 20 S
proteasome is not required for MHC class I expression and antigen
presentation (36, 37) but some viral antigens are
presented more efficiently.
How LMP2 and LMP7 mediate these effects on antigen presentation remained a controversial issue. In some laboratories in vitro experiments with fluorogenic peptides and proteasomes from an LMP2/LMP7 doubly deficient lymphoblastoid cell line yielded a reduction in cleavage at the C terminus of tyrosine and arginine residues as compared to wild type(38, 39) , whereas this was not found by other investigators(32, 40) . We have therefore readdressed this issue by strongly overexpressing LMP2 and LMP7 alone or together in transfected murine fibroblast cells. We further tested how the REG would influence these proteasome populations in in vitro digests of a 25-mer peptide. While LMP2 and LMP7 caused substantial variation in the quantity of different peptides produced, binding of the REG to any of these 20 S proteasome preparations led to a characteristic qualitative and quantitative shift in the cleavage products generated.
Figure 3: Kinetic of the degradation of a 25-mer polypeptide by 20 S proteasomes in the absence and presence of REG. A synthetic 25-mer polypeptide (sequence displayed in Fig. 4) was subjected to digest by B8 derived 20 S proteasomes in the absence of presence of an 8-fold molar excess of REG. Aliquots of the reaction mixture were withdrawn at indicated times and separated by HPLC. The most prominent peak in the profile B8 5 h is the fragment GPSEKRVWMS generated from a cleavage C-terminal of leucine.
Figure 4:
Amounts of five selected peptide fragments
produced by in vitro digest from a 25-mer polypeptide. 20 S
proteasomes isolated from B8 wild-type cells and transfectants BC2P6
(B8/LMP2), B7H6 (B8/LMP7), BC27H7 (B8/LMP2+7) were used to digest
a 25-mer peptide (sequence shown) in the presence (closed
columns) and absence (open columns) of REG. The sequences
of the five selected peptides is displayed above; note that peptide 2
is the immunodominant nonamer. Digests were performed to completion
that is for 48 h in the absence and for 24 h in the presence of REG as
described under ``Materials and Methods.'' Peptide products
were separated on HPLC and identified by their mass/charge values in
mass spectrometry. The relative amounts were derived from the ion
current shown in Table 2. The absolute amount of peptides were
calculated from the absorbance of UV light at 214 nm. For calibration
the absorbance of three synthetic peptides (4, 9, and 11 amino acids
long) were correlated with their number of peptide bonds. ,
-REG;
, +REG.
For digestion of a synthetic 25-mer
peptide derived from the sequence of Murine Cytomegalovirus pp89 IE
protein, 20 µg of peptide (kindly provided by Dr. P. Henklein,
Berlin) were dissolved in 300 µl of buffer G (30 mM Tris-HCl, pH 7.5, 10 mM KCl, 0.5 mM DTE) and
digested with 1 µg of purified proteasome for indicated times at 37
°C. REG was added at 8-fold molar excess in TEAD buffer + 1
volume of glycerol, and the negative controls were supplemented with
TEAD/glycerol buffer only. Cleavage products were analyzed by reverse
phase HPLC: 50 µl of digest was applied to a 4.6 250 mm
Ultrasphere RP18 column (Beckman) on a System Gold (Beckman) and eluted
with a flow rate of 0.5 ml/min and a linear gradient of solution A
(water, 0.1% trifluoroacetic acid) and solution B (acetonitrile, 0.1%
trifluoroacetic acid): 0-5 min 0% B, 5-40 min linear
increase to 60% B, peaks were detected at 220 nm.
A
representative clone out of the LMP2 (BC2P6), the LMP7 (B7H6), and
double transfectants (BC27P7) were raised in bulk culture, and 20 S
proteasomes were purified. Analysis of the subunit pattern on
two-dimensional NEPHGE-PAGE gels (Fig. 1) convincingly documents
the overexpression of LMP2 and LMP7 as well as an extensive replacement
of subunit by LMP2 and MB1 by LMP7, respectively. Except for this
exchange no other consistent alterations in the two-dimensional pattern
of the 20 S proteasome were noted. The significant acidic shift of the
subunit C8 (30 kDa, basic of delta) in the double transfectant might be
due to phosphorylation(45) , but as it was not observed in a
second preparation we did not further investigate this issue. The size
and isoelectric point of the overexpressed LMP2 and LMP7 proteins in
single and double transfectants are identical to those of the
endogenously expressed proteins. Overexpression of one LMP subunit does
not affect the endogenous expression of the other LMP subunit. Thus, in
contrast to what has been suggested by other
investigators(46) , in our system LMP2 and LMP7 do not need
each other nor any further IFN-
-inducible factor for incorporation
into the 20 S proteasome.
Figure 1:
Two-dimensional
NEPHGE/PAGE gels of 20 S proteasomes purified from LMP2- and
LMP7-transfected B8 cells. 20 S proteasomes were isolated from B8
wild-type cells and transfectant clones BC2P6 (B8/LMP2), B7H6 (B8/LMP7), BC27H7 (B8/ LMP2+7). We applied 50
µg of protein to each gel as detailed under ``Materials and
Methods.'' Note the exchange of LMP2 for and LMP7 for MB1
(labeled with arrows in the upper left panel) in the
respective transfectants. These preparations were used for all
subsequent experiments.
The evidence that LMP2 and LMP7 exert their effects by altering the cleavage pattern produced by the 20 S proteasome has largely been derived from experiments using short fluorogenic peptides. Using these substrates, the results reported by different investigators may differ dramatically from each other and even appear contradictory(32, 38, 39, 40, 46) . Therefore, we set up experiments to test whether LMP2 and LMP7 possibly operate via proteasome activation by the 11 S regulator. As the material for regulator isolation is very limited in mice, we chose rabbit erythrocytes as a more abundant source. When analyzed by SDS-PAGE, regulator protein purified to apparent homogeneity (and used in all subsequent experiments) is resolved into two closely migrating bands of 29 and 31 kDa (Fig. 2) which correspond to the two constituent subunits of the native regulator molecule.
Figure 2: SDS-PAGE of REG isolated from rabbit erythrocytes. Purified REG (2 µg) was subjected to SDS-PAGE on a 10-20% (w/v) acrylamide continuous gradient and electrophorized as detailed elsewhere(55) . The larger and smaller REG subunit migrate to a position corresponding to a molecular mass of about 31 and 29 kDa, respectively. Standard proteins of known molecular mass are: phosphorylase b (92.5 kDa), bovine serum albumin (67 kDa), ovalbumin (45 kDa), carbonic anhydrase (31 kDa), soybean trypsin inhibitor (21.5 kDa), and lysozyme (14.4 kDa).
In order to establish the amount of REG required for maximally stimulating 20 S proteasomes from wild-type cells and LMP transfectants, we have measured the cleavage activity toward Suc-LLVY-MCA as substrate. Maximum activation of proteasomes was achieved at an 8-fold molar excess of REG. Maximal activation by REG was about 15-fold in all proteasome preparations tested. Thus, at least in this system, there is no evidence that proteasomal LMP2/7 content affects activation factors or equilibrium constants of REG-proteasome binding. A kinetic analysis of Suc-LLVY-MCA hydrolyzing activity in the presence and absence of REG confirmed that the reduction of activity observed following overexpression of LMP2 and LMP7 (Table 1) is not compensated by binding of the REG (data not shown).
The 25-mer peptide was incubated with 20 S proteasomes purified from B8 cells in the presence or absence of an 8-fold molar excess of REG from rabbit erythrocytes. Aliquots were withdrawn at indicated times and analyzed by reverse phase HPLC. The peptide region of the HPLC profiles generated with or without REG are shown in Fig. 3. In digests where 25-mer is exposed to B8 proteasomes in the absence of REG, a time-dependent change in the cleavage profile of the peptide is observed, and only at 48 h of incubation is this profile stable. In contrast, in the presence of REG, the same effect is observed at a much earlier time, that is 10 h after starting the incubation. The intact 25-mer is primarily cleaved to yield a dominant intermediate product after 5 h of digest while in the presence of REG many additional cleavage products of comparable peak magnitude are seen. To test whether kinetic differences might be due to inactivation of proteasome or REG, aliquots were removed after 24 and 36 h of incubation and activity toward Suc-LLVY-MCA was tested. The results (not shown) revealed no loss of activity in proteasome + REG incubations, while in experiments without REG, proteasome activity was even enhanced after 24 h. As an important control the 25-mer was incubated with our REG preparation alone since it cannot be completely ruled out that the REG might be proteolytically active itself. The 25-mer was not processed after 10 h of incubation at 37 °C, and only negligible degradation is seen after 24 h. Thus, we conclude that the REG is not an active protease by itself. Our peptide profiles illustrate that the REG accelerates the digest considerably, that it strongly influences the priority of initial cleavages, and that qualitatively different peptides are generated.
Strikingly, when the REG was included in the digests, the peptide profile generated by all proteasome preparations changed in a characteristic manner. This change was quantitative as well as qualitative since several peptides were newly generated (e.g.peptide 4 in Fig. 4) while other peptides disappeared (e.g. the MYPHFMPTNL peptide in Table 2). In the presence of REG, quantitative differences between the amount of peptides produced by proteasomes with different LMP content are still observed, indicating that REG binding does not compensate LMP-mediated cleavage preferences. Again our data fail to confirm a preferential activation of cleavage after tyrosine following the binding of the REG to the 20 S proteasome as has been deduced from experiments with fluorogenic peptides. Peptides 1, 3, 4, and 5 are all generated by cleavages C-terminal of a tyrosine residue, but only peptides 4 and 5 are produced in greater amounts as a consequence of REG binding. The only consistent finding is a reduction of all peptides with a leucine at their C terminus in the presence of REG. We have confirmed the impact of REG binding on cleavage preferences by using two further synthetic 25-mer peptide substrates in additional experiments (data not shown). Taken together, these in vitro studies suggest that binding of the REG to the 20 S proteasome contributes to the diversity in generation of antigenic peptides to a similar extent as does the incorporation of LMP subunits.
In this study, we have shown that binding of the 11 S regulator markedly alters the quality and quantity of peptide products generated by the 20 S proteasome. In the same in vitro digestion assay using a 25-mer peptide as a substrate, we demonstrate that single or joint incorporation of LMP2 and LMP7 subunits into the 20 S proteasome likewise changes the quantity of different petide products generated. The REG does not preferentially activate LMP2 or 7 containing proteasomes, and it does not compensate for LMP-associated alterations in proteasomal cleavage specificity. Hence, the incorporation of LMP subunits into the 20 S proteasome and binding of the REG could both function to increase the variation of peptides produced for antigen presentation on MHC class I molecules.
When the genes encoding LMP2 and LMP7 were found to map to the MHC
class II region and shown to be IFN- inducible, the data gave an
apparently congruent picture: LMP2 and LMP7 coinduction was reported to
double the cleavage rate C-terminal of arginine and tyrosine in
fluorogenic substrates, and it was suggested that this generates more
peptides which meet the binding requirements of MHC class I molecules (38, 39) . Recently, this view has been challenged by
Ustrell et al.(40) who find no significant impact of
LMP subunits on the peptidase activity of purified 20 S proteasomes or
by Boes et al.(32) who reported that
IFN-
-mediated incorporation of LMP2 and LMP7 reduces rather than
enhances the cleavage C-terminal of tyrosine residues. The data
presented in Table 1are in accordance with the findings of Boes et al. in that combined incorporation of LMP2 and LMP7 reduced
the Suc-LLVY-MCA hydrolyzing activity by about 40%. The transfection
approach allowed us to test if single incorporation of either LMP2 or
LMP7 had any effect on the cleavage of fluorogenic peptides. This was
clearly the case: incorporation of LMP2 alone, for example, reduced the
cleavage of the (Z)-LLE-
NA by about 50%. Thus, our data
obtained with fluorogenic peptides support the concept that
incorporation of the LMP subunits does alter the cleavage
characteristics of the 20 S proteasome. It is quite difficult to
rationalize why other laboratories performing similar in vitro experiments obtain controversial results. We do not feel that
these discrepancies can be attributed to the use of different cell line
models, as we obtained identical results with B8 mouse fibroblast
cells, T2 lymphoblastoid cells, or Sci/ET27F pre-T cells(51) ,
and identical results were obtained with IFN-
stimulation or
LMP2/LMP7 double transfection. A major cause of discrepancy might be
the different purification protocols of 20 S proteasomes applied by
different laboratories. We noticed that following our protocol a high
molecular weight protease complex copurifies with the 20 S proteasome
and is only separated in the last step by MonoQ chromatography. This
complex is not related to the 20 S proteasome as it does not react with
anti-proteasome antibodies in Western blots (data not shown), but it
has protein chemical properties similar to
-
macroglobulin-cathepsin complexes as described by Dahlmann et
al.(52) . This complex hydrolyzes fluorogenic arginine and
tyrosine but not leucine substrates, and if a purification scheme does
not remove this complex, the results will not be comparable to those
obtained with our protocol. We performed our peptidase assays with
freshly isolated proteasomes which is important because we noticed that
the peptidase activity may be changed when the complex was frozen. This
is a further source of controversy between different laboratories.
The sequence of our model 25-mer polypeptide is derived from the
immediate early protein pp89 of the murine cytomegalovirus. It contains
the nonamer YPHFMPTNL which is an immunodominant T cell epitope for the
presentation on H-2L MHC class I molecules. In previous
experiments it was not possible to directly measure the production of
this nonamer in vitro(32) . With the help of
electrospray mass spectrometry, we were able to identify and quantify
the production of this nonamer peptide (peptide 2 in Fig. 4):
the highest amount was detected in digests conducted with proteasomes
from B8 wild-type cells and the LMP2 transfectant whereas LMP7
overexpression had an adverse effect. Binding of the REG to the 20 S
proteasome markedly reduced the amount of nonamer, and the reduction
was least prominent in the case of the LMP2 transfectant. For the
generation of this nonamer, the presence of the REG appears to be a
disadvantage, whereas the production of other nonamer peptides might be
favored. Apparently, the immune system varies at the level of
populations by maintaining a polymorphism in MHC haplotypes with
different anchor residues, at the level of single cells though, by
variation in antigen processing. For this purpose the immune system may
have recruited the REG. Not only binding to the proteasome itself but
also the level of incorporation of either of the 29- and 31-kDa
subunits into the ringshaped 11 S regulator might contribute to
diversity in antigen processing. Apart from antigen presentation it
might not be arbitrary which peptides are being generated during the
degradation of cytosolic proteins for intracellular communication.
A
puzzling question remains: how does a viral protein find an access to
the interior of the 20 S proteasome? In vitro, the 20 S
proteasome does not cleave intact proteins, with very few known
exceptions, and binding of the REG apparently does not change this
property of the 20 S proteasome(20, 21) . Earlier data
obtained with a cell line mutant, partially deficient in ubiquitin
activation, had suggested that ubiquitin conjugation and degradation
via the 26 S proteasome might be essential to antigen
presentation(53) , but as a subsequent report by other
investigators has raised doubts on this pathway(54) , the
question remains unresolved. Theoretically, it would be conceivable
that the REG binds to one -end plate of the 20 S proteasome and a
19 S regulator complex to the opposite
-end plate, thus accepting
ubiquitinated proteins. However, in the light of experimental data
reported by Hoffmann and Rechsteiner (28) such a possibility
seems unlikely, and further research will be required to unravel these
questions.