The JI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Frisan, T.
Right arrow Articles by Masucci, M. G.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Frisan, T.
Right arrow Articles by Masucci, M. G.
The Journal of Immunology, 1998, 160: 3281-3289.
Copyright © 1998 by The American Association of Immunologists

Phenotype-Dependent Differences in Proteasome Subunit Composition and Cleavage Specificity in B Cell Lines1

Teresa Frisan2,*, Victor Levitsky*, Axel Polack{dagger} and Maria G. Masucci*

* Microbiology and Tumor Biology Center, Karolinska Institute, Stockholm, Sweden; and {dagger} Istitut für Klinische Molekularbiologie und Tumorgenetik, Gesellschaft für Strahlung und Umweltforschung-Forschungszentrum für Umwelt und Gesundheit, Munich, Germany


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We have compared the subunit composition and enzymatic activity of purified 26S proteasomes from Burkitt’s lymphoma (BL) cells and in vitro EBV-transformed lymphoblastoid cell lines (LCLs) of normal B cell origin. Low expression of the IFN-{gamma}-regulated ß low molecular mass polypeptide (Lmp)2, Lmp7, and MECL-1 was demonstrated in a panel of seven BL lines that express the germinal center cell phenotype of the original tumor. Coexpression of Lmp2 and Lmp7 with the constitutively expressed subunits {delta} and MB1 was demonstrated in the BL lines by immunoprecipitation and two-dimensional gel fractionation of the 20S proteasomes. Coexpression of these subunits correlated with reduced levels of chymotrypsin- and trypsin-like activities detected by the cleavage of fluorogenic substrates. Down-regulation of Lmp2 and Lmp7 and decreased chymotrypsin- and trypsin-like activities were also observed in purified proteasomes from a c-myc-transfected subline of the ER/EB2–5 LCL that has adopted a BL-like phenotype. A synthetic peptide analogue of the immunodominant epitope from the EBV nuclear Ag 4 (E4416–424Y) was cleaved by proteasomes from BLs and A1, while proteasomes from LCLs were inactive. Cleavage of the E4416–424Y peptide was not affected by treatment of the BL cells with IFN-{gamma} despite both significant up-regulation of Lmp2 and Lmp7 and reconstitution of chymotrypsin and trypsin-like activities against fluorogenic substrates to LCL-like levels. The results demonstrate that B cell lines representing different stages of B cell activation and differentiation express proteasomes with different subunit compositions and enzymatic activity. This may result in the generation of a distinct set of endogenous peptides and influence the immunogenicity of these cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The presentation of endogenously expressed protein Ags to CTLs requires degradation in the cytosol and transport of the generated peptides into the lumen of the endoplasmic reticulum (ER)3 by a heterodimeric complex known as TAP. In the ER, the peptides associate with newly synthesized class I heavy chains and ß2-microglobulin (ß2m) to form stable trimolecular complexes that are subsequently transported to the cell surface (reviewed in 1 . Recent evidence suggests that the multicatalytic protease complex known as the proteasome participates in the generation of antigenic peptides for MHC class I-restricted presentation (2, 3).

The proteasome is composed of a 20S cylindrical proteolytic core that binds activator complexes designated 19S regulator and 11S regulator (or PA28) at its ends. The 19S regulator associates with the 20S proteasome to form the 26S protease, which is involved in the targeted degradation of ubiquitin-conjugated proteins (reviewed in 4 . The 20S core consists of 14 subunits ranging in molecular mass from 21 to 32 kDa (reviewed in 5 . The subunits are classified as {alpha} and ß type based on their homology to the subunits of an ancestral proteasome found in the archaebacterium Thermoplasma acidophilum (6). The active proteolytic sites are contained in the ß subunits that constitute the two inner rings of the catalytic core (7). The genes encoding for two ß subunits, designated Lmp2 and Lmp7, have been mapped to the MHC class II locus (8, 9, 10). Lmp2 and Lmp7 are induced by treatment with IFN-{gamma} and replace the two constitutively expressed subunits, {delta} and MB1 (11). A third subunit exchange has been detected following IFN-{gamma} treatment of mouse fibroblasts; the non-MHC-encoded MECL-1 subunit was shown to replace the thus far poorly characterized ß subunit MC14 (12, 13).

The proteasome cleaves peptide bonds after hydrophobic (chymotrypsin-like activity), basic (trypsin-like activity), and acidic (peptidylglutamyl peptide-hydrolyzing) residues (reviewed in 14 , but very little is known about the contribution of the various ß subunits to this enzymatic activity. Recent evidence suggests that incorporation of low molecular mass polypeptide (Lmp)2 and Lmp7 in the 20S proteasome core following IFN-{gamma} treatment may alter the peptidase activity, favoring cleavage at the carboxyl side of hydrophobic and basic residues (15, 16). It is not known whether additional differences in the expression of catalytic or regulatory subunits may occur as a result of cell type and/or state of activation.

Evasion from CTL-mediated rejection is likely to be a critical step in tumor development. The EBV-associated Burkitt’s lymphoma (BL) is a classical example of a human tumor that evades CTL surveillance. The BL tumor cells are phenotypically similar to germinal center centroblasts/centrocytes with high expression of CD10 and CD77 and a lack of adhesion molecules and activation markers such as CD23 and CD39 (17). BL-derived cell lines are classified into three major groups that differ in cell phenotype and pattern of growth in vitro. Group I BL lines grow as a single-cell suspension and have retained the surface marker expression of the original tumor cells. Group II cell lines are characterized by coexpression of CD10 and activation markers, up-regulation of adhesion molecules, and growth in large clumps. BL lines expressing a group III phenotype closely resemble EBV-transformed lymphoblastoid cell lines (LCLs) and have lost expression of CD10 and BLA (reviewed in 18 . The viral gene expression of EBV-positive group I BL lines is restricted to the EBV nuclear Ag (EBNA)1, while drift to a group III phenotype is accompanied by expression of six nuclear Ags (EBNA1 to EBNA6) and three latent membrane proteins ((LMP)1, -2A, and -2B) (19). EBV-positive BL lines that express a group I/II phenotype are not lysed by EBV-specific CTLs (20). Recent evidence suggests that several steps in the Ag-processing and -presentation pathway may be impaired in these cells. Down-regulation of the TAP-1 and TAP-2 transporters was demonstrated in BL lines that failed to present CTL epitopes derived from EBNA3 (21). Lysis was partially restored by transfection of a minigene encoding the epitope linked to an ER localization sequence (21) or by transactivation of the TAP-1 gene following LMP1 up-regulation/transfection (22). However, the demonstration that full reconstitution of TAP activity did not restore presentation of an HLA A11-restricted epitope derived from EBNA4 (23) suggests the presence of an additional defect(s) located upstream of peptide transport and MHC class I assembly and maturation.

We have now compared the subunit composition and cleavage specificity of purified proteasomes derived from BL lines expressing a group I/II phenotype, in vitro EBV-transformed LCLs that constitutively express a group III phenotype, and an LCL that has acquired BL-like characteristics due to constitutive expression of transfected c-myc and down-regulation of the EBV-encoded EBNA2 and LMP1. We demonstrate that the BL cell phenotype is associated with lower expression of three IFN-{gamma}-inducible ß subunits and different cleavage specificity of the proteasome. Thus, a characteristic set of antigenic peptides may be produced in these cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell lines

BL28 was established from an EBV-negative tumor biopsy (24), while Akata (25), BL72 (24), and WW1-BL (26) were established from EBV-positive tumor biopsies. The BL28 and Akata cell lines have been classified as group I BLs on the basis of surface marker and EBV Ag expression, while BL72 and WW1-BL express a group II phenotype. The DH-LCL was established by spontaneous outgrowth from the lymphocytes of a normal EBV-seropositive donor cultured in the presence of 0.1 µg/ml cyclosporin A. This cell line carries an EBV strain that lacks the immunodominant HLA A11-restricted epitope E4416–424 (27). The QJZ-LCL, JAC-LCL and EA-LCL were obtained by in vitro infection of normal B lymphocytes with the B95.8 strain of EBV. The TAP-1- and -2-deficient and Lmp2- and LMP7-deficient T2 line (28) was used as a control in the analysis of Lmp protein expression by Western blot and two-dimensional (2D) gel fractionation. The ER/EB2–5 LCL expresses a conditional mutant of the EBNA2 gene that requires 1 µM ß-estradiol (Sigma, St. Louis, MO) for activation of protein function (29). The A1 subline was obtained by stable transfection of a c-myc expression plasmid as described (30). All lines were cultured in RPMI 1640 (Life Technologies, Grand Island, NY), supplemented with 2 mM glutamine, 100 U/ml penicillin, 100 U/ml streptomycin, and 10% heat-inactivated FCS. Where indicated, the cell lines were cultured for 48 h in medium containing 500 international U/ml of IFN-{gamma} before the assays.

Materials

The proteasome inhibitors lactacystin and carbobenzoxy-L-leucyl-L-leucyl-L-leucinal (z-LLL) were purchased from Calbiochem (La Jolla, CA) and Peptides International (Louisville, KY), respectively. The protease inhibitors leupeptin, pepstatin, and bestatin were purchased from Boehringer Mannheim (Mannheim, Germany). The calpain inhibitor N-acetyl-L-leucinyl-L-leucinal-L-norleucinal (LLnL) was purchased from Sigma. A synthetic analogue of the EBNA4 epitope E4416–424 (IVTDFSVIK) with a Phe'Tyr substitution in position 5 (E4416–424Y) was synthesized by the Merrifield solid-phase method (31) (Alta Bioscience, Birmingham, U.K.). HPLC-purified peptide was dissolved in PBS at a concentration of 0.5 mg/ml and stored at -20°C.

Western blotting

The equivalents of 2 x 105 cells were fractionated by SDS-PAGE in denaturing 12% acrylamide gels and blotted onto nitrocellulose filters according to standard procedures (32). The blots were probed with Lmp2, Lmp7, MECL-1, and {delta}-specific rabbit sera (obtained from Drs. J. Trowsdale and I. Correa) Division of Immunology; Department of Pathology University of Cambridge, Cambridge, U.K. (33) at a 1:1000 dilution or with a 1:50 dilution of the mouse mAb MCP21 (a kind gift of Dr. K. B. Hendil) August Krogh Institute; University of Copenhagen, Copenhagen, Denmark. Expression of the EBV Ags EBNA2 and LMP1 was assayed using a 1:20 dilution of a previously characterized human serum HR containing high Ab titers to all EBNAs (viral capsid Ag, 1:640; EBNA1, 1:80; EBNA2A, 1:160; EBNA2B, 1:80; EBNA6, 1:320) and a 1:10,000 dilution of the LMP1-specific mAb S-12 (34). The c-Myc protein was detected using the mAb 9E10 (a kind gift of Dr. G. Evan) Imperial Cancer Research Fund (ICRF), London, U.K. at 1:500 dilution. The blots were reacted with horseradish peroxidase-labeled anti-rabbit, anti-human, or anti-mouse Abs, and the immunocomplexes were visualized by enhanced chemiluminescence according to the manufacturer’s instructions (Amersham, Little Chalfont, U.K.).

2D Gel electrophoresis

Five x 106 cells were metabolically labelled by culture for 5 h at 37°C in 2 ml of methionine-free RPMI 1640 medium supplemented with 10% FCS (Life Technologies) and 100 µCi of [35S]methionine (Amersham). The cells were lysed for 30 min at 4°C in a buffer containing 50 mM Tris-HCl (pH 7.5), 5 mM MgCl2, 5 mM EDTA, 0.5% Nonidet P-40, and 1 mM PMSF, and the nuclei were pelleted by centrifugation at 15,000 g. The supernatants were transferred to a clean Eppendorf tube and precleared by incubation with 2 µl mouse serum for 1 h at 4°C on a rocking platform, followed by 2x incubation with 50 µl of a 10% suspension of formalin-fixed Staphylococcus aureus (Sigma) in NET buffer (50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 5 mM EDTA, 0.5% v/v Nonidet P-40). Immunoprecipitation with the mAb MCP21 (35) was performed from equal amounts of TCA-precipitable material for 3 h at 4°C, and the immunocomplexes were recovered with 50 µl of S. aureus for 2 h at 4°C. After three washes in NET buffer and a final wash in 50 mM ammonium acetate (pH 7), the pellet was resuspended in 1.5 ml of distilled water and lyophilized. The immunoprecipitates were dissolved in 50 µl of nonequilibrium pH gel electrophoresis (NEPHGE) buffer (9 M urea, 5% mercaptoethanol, 2% Triton X-100, 2.5% Pharmalyte 3–10, 0.8% Pharmalyte 2.5–5) (Pharmacia, Uppsala, Sweden) and incubated 3 to 4 h at room temperature. Twenty-five microliters were then subjected to NEPHGE using the MiniProtean II cell (Bio-Rad, Hercules, CA). Briefly, gel rods for the first dimension were prepared in 1 mm x 7.5-cm glass tubes by adding 18.75 µl 10% ammonium persulfate and 6.25 µl N,N,N',N'-tetramethylethylenediamine to a degassed solution of 2.63 g of urea, 625 µl acrylamide stock solution (29.2% acrylamide and 0.8% bisacrylamide), 312 µl Pharmalyte 3–10, 100 µl Pharmalyte 2.5–5, 94 µl Triton X-100, and 1.8 ml MilliQ H2O. After polymerization for 1 h, the samples were applied to the gel, overlaid with 60 µl of NEPHGE buffer, and run for 4 h at 400 V from the anode (0.01 M H3PO4) to the cathode (0.02 M NaOH). The second dimension was run on denaturing 12.5% acrylamide gels. The gels were dried, exposed to a PhosphorImager screen for 24 h (LCLs) or 48 h (BLs), and then densitometric analysis was performed using the Image Quant software (Molecular Dynamics, Sunnyvale, CA).

Purification of the 26S proteasome complex

26S proteasomes were purified from 2 x 108 cells according to the protocol of Driscoll et al. (16). Briefly, frozen cell pellets were resuspended in a buffer containing 20 mM Tris-HCl (pH 7.5), 2 mM MgCl2, 0.1 mM EDTA, 2 mM ATP, and 1 mM DTT, and membranes were broken by 50 strokes in a Dounce homogenizer. The nuclei were removed by centrifugation at 15,000 g for 15 min, and the supernatant was further centrifuged at 100,000 g for 60 min. The 100,000-g supernatant was applied to a Mono-Q (HR 5/5) column and fractionated by fast protein liquid chromatography (FPLC) (Pharmacia). Bound proteins were eluted with an NaCl gradient of buffer A (20 mM Tris-HCl, pH 7.2) and buffer B (20 mM Tris-HCl, pH 7.2, 1 M NaCl) as follows: for 0 to 10 min, 100% buffer A at a flow rate of 1 ml/min; for 10 to 40 min, linear increase to 34% buffer B at a flow rate of 1 ml/min; for 40 to 100 min, linear increase to 60% buffer B at a flow rate of 0.5 ml/min; and for 100 to 110 min, linear increase to 100% buffer B at a flow rate of 1 ml/min. Eighty 1 ml fractions were collected. The peptidase activity of each fraction was determined fluorometrically, and proteasome-containing fractions were pooled, supplemented with 10% (v/v) glycerol and 2 mM ATP, and stored at -70°C in 100 µl aliquots. The purity of the proteasome fractions was assessed by SDS-PAGE and silver staining (36). The protein concentration of the active fractions was measured by the BCA Protein Assay Reagent Kit (Pierce, Rockford, IL).

Enzyme assays

The fluorogenic peptides N-Succinyl-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin (SucLLVY-MCA) and N-tert-butoxycarbonyl-Leu-Arg-Arg-7-amino-4-methylcoumarin (BocLRR-MCA) were used to assess proteasome activity. The rates of cleavage by purified proteasomes were analyzed with different substrate concentrations at 37°C for 1 h. The reactions were quenched using cold ethanol, and fluorescence was determined in a luminescence spectrometer (Perkin-Elmer, Beaconsfield, U.K.) using an excitation of 380 nm and emission of 440 nm. The Vmax for each proteasome preparation was calculated according to the Lineweaver-Burk equation (37) and expressed as nmol of substrate degraded per µg of enzyme per h.

Peptide degradation assays

The E4416–424Y peptide was 125I-labeled by chloramine T-catalyzed iodination (38). Aliquots (100 µl) of the proteasome preparation were desalted by ultrafiltration (Microcon-10, Amicon, Beverly, MA), and 1 µg of enzyme was incubated with 30 ng of iodinated peptide at 37°C for the indicated time in 30 µl of assay buffer (20 mM Tris-HCl (pH 7.5), 2 mM MgCl2, 0.1 mM EDTA and 1 mM DTT). The degradation products were resolved in 7 x 8 cm x 1 mm tricine gels (39). Dried gels were exposed to a PhosphorImager screen and analyzed with the Image Quant software (Molecular Dynamics). Where indicated, the proteasomes were preincubated with lactacystin for 20 min at 37°C before addition of the substrate.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Expression of the IFN-{gamma}-regulated ß subunits

The levels of expression of the IFN-{gamma} regulated ß subunits Lmp2, Lmp7, and MECL-1 were compared by Western blot analysis in both a panel of BL lines that have retained the phenotypic characteristics of the original tumor and in regular in vitro EBV-transformed LCLs of normal B cell origin. A representative experiment, including two group I lines (Akata and BL28), two group II lines (WW1-BL and BL72), and two reference LCLs, is shown in Figure 1Go. A significant down-regulation of Lmp2 and a less marked down-regulation of Lmp7 and MECL-1 was detected in all four BL lines compared with LCLs. Similar results were obtained in three additional group I BLs: DG75, BL41, and Rael (data not shown). In repeated experiments, the expression of Lmp2 appeared to be somewhat lower in group I compared with group II BLs. All the cell lines contained similar amounts of proteasomes as judged by the detection of comparable levels of the constitutively expressed {alpha} subunit MC6.



View larger version (76K):
[in this window]
[in a new window]
  		FIGURE 1. Expression of Lmp2, Lmp7, MECL-1, and MC6 subunits in BL cells. Lysates from 2 x 105 LCL and BL cells were separated by SDS-PAGE in a 12% resolving gel and transferred to nitrocellulose filters. The blots were probed with rabbit sera specific for human Lmp2, Lmp7 and MECL-1 and were also probed with the mAb MCP21 that recognizes the {alpha} subunit MC6.

 
Proteasome subunit composition

The proteasome subunit composition was further investigated by immunoprecipitation from metabolically labeled cells and by 2D gel fractionation. At least two independent experiments were performed for each cell line tested. The various proteasome subunits were identified by comparison with published data (12, 35) and, when specific Abs were available, by Western blot analysis of the 2D gels. Representative experiments illustrating the results obtained with two BL lines, BL28 and BL72, and one of four LCLs are shown in Figure 2Go. The ß subunits Lmp2 and Lmp7 were easily recognized in proteasomes derived from LCLs, while the corresponding {delta} and MB1 subunits were either poorly expressed or, within the limit of sensitivity of our detection system, appeared to be absent (Fig. 2GoA). {delta} and MB1 were clearly coexpressed with Lmp2 and Lmp7 in BL cells (Fig. 2Go, B and C). The relative representation of the Lmp2/{delta} and Lmp7/MB1 pairs was evaluated by densitometry scanning of the autoradiograms. A compilation of the results of this analysis is shown in Table IGo. A {delta}/LMP2 ratio of >1 was observed in all experiments performed with BL cells, while the intensity of the MB1 spot was ~10-fold lower compared with LMP7. The {delta}/LMP2 and MB1/LMP7 ratios were at least 10-fold lower in LCLs.



View larger version (118K):
[in this window]
[in a new window]
  		FIGURE 2. Subunit composition of BL and LCL proteasomes. The proteasome complex was immunoprecipitated from 35S-labeled QJZ-LCL, BL28, and BL72 cells and further fractionated by two-dimensional PAGE with NEPHGE in the first dimension and SDS-PAGE in the second dimension. The various subunits were identified in gels exposed for 24 h (LCLs) or 48 h (BLs) on the basis of their isoelectric point by comparison with published data (12, 35) and by Western blot analysis when specific Abs where available. The position of the Lmp2, {delta}, Lmp7, and MB1 subunits is indicated.

 

View this table:
[in this window]
[in a new window]
  		Table I. Relative expression of constitutive and IFN-{gamma}-inducible ß subunits in LCLs and BLs1

 
Proteasome purification and enzymatic activity

To investigate whether the differences in subunit composition detected by Western blot and 2D gel analysis correlated with differences in enzymatic activity, we have analyzed the cleavage specificity of 26S proteasomes isolated from LCL and BL cells. A representative experiment illustrating the FPLC purification procedure and the enzymatic activity of proteasomes from the DH-LCL is shown in Figure 3Go. The chromatographic profile at 280 nm of fractions 36 to 57 is presented in panel 3A. Each fraction was assessed for proteolytic activity against the fluorogenic substrate SucLLVY-MCA. One single activity peak was identified between fractions 48 and 52 corresponding to a single A280 peak eluted at 42% buffer B. The cleavage of the SucLLVY-MCA substrate was inhibited in a dose-dependent manner by the specific proteasome inhibitor lactacystin (Fig. 3GoB) and by other proteasome inhibitors such as z-LLL and LnLL but not by the aminopeptidase specific inhibitor bestatin (data not shown), confirming that the enzymatic activity was associated with the proteasome complex. Furthermore, Lmp2 and Lmp7 were detected in these fractions by Western blot analysis (data not shown). The purity of the proteasome preparations was confirmed by SDS-PAGE fractionation and silver staining that revealed the presence of closely migrating polypeptides with a molecular mass ranging from 20 to 32 kDa and additional bands with a molecular mass of between 30 and 110 kDa (data not shown). A similar migration pattern was reported for purified 26S proteasome from rabbit reticulocytes (40).



View larger version (24K):
[in this window]
[in a new window]
  		FIGURE 3. Purification and functional characterization of the 26S proteasome complex. 26S proteasomes were purified from cytosolic cell extracts by FPLC fractionation as described in Materials and Methods. A, Chromatogram profile of fractions 36 to 57 in one representative experiment performed with the DH-LCL. Bound proteins were eluted from the column with an NaCl gradient (–). The eluted proteins were detected by monitoring UV absorbance (A280), and the peptide-hydrolyzing activity (•) was assayed as described using 10 µl of each fraction. B, Effect of the proteasome inhibitor lactacystin on the degradation of the SucLLVY-MCA substrate. One microgram of enzyme was incubated for 20 min at 37°C with the indicated concentration of the inhibitor. The substrate was then added to the reaction mixture and fluorescence was measured after an incubation for 1 h at 37°C. C, Enzyme kinetics analysis of proteasome activity on the SucLLVY-MCA and BocLRR-MCA substrates. One microgram of enzyme was incubated with an increasing substrate concentration (25–200 µM). The cleavage activity was evaluated as fluorescence units and is expressed as nmol of substrate degraded per µg of enzyme per h. The Vmax values are 111 nmol/µg/h for SucLLVY-MCA and 100 nmol/µg/h for BocLRR-MCA.

 
The activity of the purified proteasome preparations was estimated in enzyme kinetics assays (Fig. 3GoC). One microgram of the enzyme was incubated with an increasing concentration (25–200 µM) of the SucLLVY-MCA (chymotrypsin-like) and BocLRR-MCA (trypsin-like) substrates for 1 h at 37°C, and cleavage was evaluated as fluorescence units. The Vmax was calculated according to the Lineweaver-Burk equation (37) and is expressed as nmol of substrate degraded per µg of enzyme per h. Proteasomes purified from BL cells were tested for chymotrypsin- and trypsin-like activities in parallel with proteasome preparations derived from LCLs and from the Lmp2/7-deficient cell line T2. The results of four to eight experiments performed with two proteasome preparations from BL28, BL72, three LCLs, and T2 are summarized in Figure 4Go. The maximal activity of the BL-derived proteasomes was ~20 nmol/µg/h for SucLLVY-MCA and between 20 and 30 nmol/µg/h for BocLRR-MCA. Comparable values were obtained in a more restricted set of experiments performed with purified proteasomes from BL41 and DG75 (data not shown). This activity was similar to that of T2-derived proteasomes (23 nmol/µg/h and 13 nmol/µg/h, respectively) and was four- to sixfold lower than the activity of proteasomes derived from LCLs.



View larger version (13K):
[in this window]
[in a new window]
  		FIGURE 4. Enzymatic activity of proteasomes from BLs, LCLs, and T2 cells. The Vmax for the SucLLVY-MCA and BocLRR-MCA substrates was evaluated as described in Figure 3Go. The mean ± SD of four to eight experiments performed with two proteasome preparations from each cell line is shown.

 
Correlation between cell phenotype and proteasome activity

BL lines that have maintained the phenotypic characteristic of the original tumor resemble germinal center centrocytes/centroblasts, while group III BLs and LCLs have the phenotype of activated B blasts. To assess whether different stages of B cell activation/differentiation are associated with changes in subunit composition and enzymatic activity of the proteasome, we have used the ER/EB2–5 LCL and its c-myc transfected subline A1 as a model. ER/EB2–5 has been obtained by in vitro transformation of B lymphocytes with a recombinant EBV that carries an EBNA2-estrogen receptor fusion gene (29). Withdrawal of estrogen from the culture medium results in down-regulation of EBNA2 and LMP1, loss of the activated phenotype, and growth arrest. Expression of a constitutively activated c-myc gene drives cell proliferation in the A1 transfectant in the absence of EBNA2 and LMP1 (Fig. 5GoA) and allows the expression of a stable BL-like phenotype (30). Lmp2 and Lmp7 were strongly down-regulated in the A1 subline compared with the parental ER/EB5–2 LCL (Fig. 5GoA). Proteasomes purified from these cells exhibited decreased chymotrypsin- and trypsin-like activities and resembled those derived from authentic BL cells and the T2 cell line (Fig. 5GoB).



View larger version (16K):
[in this window]
[in a new window]
  		FIGURE 5. Proteasome subunit composition and enzymatic activity of the ER/EB2–5 and A1 cell lines. A, Western blot analysis. Total extracts from 2 x 105 cells were fractionated by SDS-PAGE, transferred to nitrocellulose filters, and probed with the c-myc-specific mouse mAb 9E10, the EBV Ab-positive polyclonal human serum HR, the LMP1-specific mouse mAb S-12, and the rabbit sera specific for human Lmp2 and Lmp7. B, Enzyme kinetics analysis of proteasomes from ER/EB2–5 and A1 cells. The Vmax for the SucLLVY-MCA and BocLRR-MCA substrates were calculated as described in Figure 3Go. The mean ± SD of four experiments is shown.

 
Cleavage of the E4416–424Y peptide

We have previously shown that HLA A11-positive BL cells fail to present the A11-restricted epitope E4416–424. The defect could not be overcome by expression of a preformed epitope even after full reconstitution of TAP-1/TAP-2 activity, suggesting that the exogenous peptide may be degraded before entering the ER lumen (23). To investigate whether the proteasome may be involved in this degradation, an analogue of the E4416–424 peptide that carries a Phe'Tyr substitution in position 5 to allow iodination (E4416–424Y) was used as substrate for degradation by proteasomes from LCLs, BLs, and the ER/EB2–5-A1 pair. As illustrated by the representative experiment shown in Figure 6Go, proteasomes purified from QJZ-LCL (Fig. 6GoB) and ER/EB2–5 (Fig. 6GoD) either did not cleave or only marginally cleaved the E4416–424Y peptide. Similar results were obtained in repeated experiments performed with proteasome preparations derived from two additional LCLs. In contrast, the E4416–424Y peptide was cleaved by proteasome derived from BL28 (Fig. 6GoA), A1 (Fig. 6GoC), and BL72 (data not shown). Two major degradation products are indicated in Figure 6Go as a and b. The a product migrated slightly faster in tricine gels compared with the uncleaved peptide, while the electrophoretic mobility of the b product was considerably higher. The appearance of these degradation products was inhibited in a dose-dependent manner by the specific proteasome inhibitors lactacystin (Fig. 6Go, A–D) and z-LLL (data not shown), but not by other protease inhibitors such as aprotinin, leupeptin, bestatin, and pepstatin (data not shown).



View larger version (90K):
[in this window]
[in a new window]
  		FIGURE 6. Degradation of the E4416–424Y peptide. Iodinated peptide (30 ng) was incubated with 1 µg of enzyme at 37°C for 12 h, and the degradation products were fractionated by tricine gels. A, BL28; B, QJZ-LCL; C, A1; D, ER/EB2–5. The positions of the undigested peptide (pep) and two major degradation products, a and b, are indicated. Similar levels of the constitutively expressed {alpha} subunit MC6 were detected by Western blot analysis of the proteasome preparations, confirming the fact that equal amounts of enzyme were used for each cell line (data not shown). Where indicated, the proteasomes were preincubated with an increasing concentration (50, 100, and 150 µM) of the specific inhibitor lactacystin (Lcy) for 20 min at 37°C before addition of the substrate.

 
Effect of IFN-{gamma}

IFN-{gamma} up-regulates the expression of several ß subunits (11, 12, 13, 41) and modulates the proteolytic activity of human and mouse 20S proteasomes (15, 16, 41). Therefore, we have investigated the effects of IFN-{gamma} on Lmp2/7 expression and on the enzymatic activity of the BL proteasomes. Representative experiments performed with the BL28 and BL72 cell lines are shown in Figure 7Go. Treatment of the BL lines for 48 h in medium containing 500 U/ml of IFN-{gamma} resulted in a strong up-regulation of Lmp2 (a 14-fold increase in BL28 and a 5-fold increase in BL72), and a two- to threefold increase was observed for Lmp7 (Fig. 7Go, A and B). A concomitant down-regulation of the {delta} subunit was also observed (Fig. 7Go, A and B). This change in subunit composition correlated with enhanced cleavage of the SucLLVY-MCA and BocLRR-MCA fluorogenic substrates (Fig. 7GoC) corresponding to chymotrypsin- and trypsin-like activities similar to those observed in LCLs (cf Fig. 4Go and Fig. 7GoC). As illustrated by the representative experiment shown in Figure 8Go, treatment with IFN-{gamma} did not influence the ability of the BL proteasomes to cleave the E4416–424Y peptide. This is in line with the inability of BL cells treated with IFN-{gamma} to present the EBNA4 Ag (23).



View larger version (17K):
[in this window]
[in a new window]
  		FIGURE 7. Effect of IFN-{gamma} on Lmp2 and Lmp7 expression and cleavage of fluorogenic substrates. A, Western blot analysis. Lysates from 2 x 105 DH-LCL, T2, and untreated or IFN-{gamma}-treated BL cells were separated by SDS-PAGE in a 12% resolving gel and transferred to nitrocellulose filters. The blots were probed with rabbit sera specific for human Lmp2, Lmp7, and {delta}. The intensity of the specific bands was measured by densitometry, and the corresponding OD is shown at the bottom of each lane. One representative experiment of four is shown. B, Expression of Lmp2 and {delta} in untreated and IFN-{gamma}-treated BL28 and BL72. C, Enzyme kinetics analysis of proteasomes from IFN-{gamma}-treated and untreated BLs. Proteasomes were purified from untreated BL cells and from cells treated for 48 h in a medium containing 500 U/ml of IFN-{gamma}. The Vmax for the SucLLVY-MCA and BocLRR-MCA substrates is shown. The mean ± SD of three to six experiments is also shown.

 


View larger version (48K):
[in this window]
[in a new window]
  		FIGURE 8. Effect of IFN-{gamma} treatment on the degradation of the E4416–424Y peptide. A, Degradation assays were performed for 12 h with proteasomes purified from untreated and IFN-{gamma}-treated BL28 cells as described in Figure 6Go. One representative experiment of three is shown. B, The intensity of the bands corresponding to the E4416–424Y degradation products was quantitated by densitometry, and the percentage of each product was calculated relative to the total intensity of each lane.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Previous results have suggested that defects in the production of antigenic peptides for MHC class I assembly and presentation may contribute to the immunoescape of BL cells (21, 22, 23). We have investigated this phenomenon by comparing the subunit composition and enzymatic activity of proteasomes derived from BL cells and from EBV-transformed LCLs of normal B cell origin.

A significant down-regulation of the ß subunit Lmp2 and a less marked down-regulation of Lmp7 and MECL-1 were demonstrated in a panel of seven BL cell lines expressing a group I or group II BL phenotype (Fig. 1Go and data not shown). The different subunit composition of proteasomes from BLs and LCLs was further confirmed by immunoprecipitation and 2D gel fractionation of the 20S proteasome complexes (Fig. 2Go). The ß subunits {delta} and MB1 were clearly coexpressed with the IFN-{gamma}-inducible Lmp2 and Lmp7 in BL cells but were expressed at very low levels or were not detected in LCLs. Since the presence of Lmp2/{delta} and Lmp7/MB1 appears to be mutually exclusive (11), the coexpression of these subunits in BLs is likely to reflect the presence of structurally different subsets of proteasomes. This possibility is in line with the findings of Brown et al. that demonstrate the existence of at least four types of proteasomes in a mouse macrophage cell line (42); only one subset of the proteasomes contained Lmp2. The significance of our findings is further confirmed by the strong down-regulation of Lmp2 and Lmp7 in the A1 transfectant that has shifted toward a BL-like phenotype due to constitutive expression of c-myc and down-regulation of EBNA2 and LMP1 (30) (Fig. 5GoA). The difference between ER/EB2–5 and A1 suggests that the distinct pattern of proteasome ß subunit composition of BLs may be a property of the particular stages of B cell activation/differentiation represented by the tumor rather than the result of in vivo selection for a nonimmunogenic phenotype.

Controversial data exist concerning the influence of Lmp2 and Lmp7 on the degradation of fluorogenic substrates and the relevance of this activity for the production of antigenic peptides in vivo. Gaczynska et al. (15) and Driscoll et al. (16) have shown that incorporation of these subunits in the proteasome complex enhances the chymotrypsin- and trypsin-like activities, while the peptidylglutamyl hydrolyzing activity was slightly diminished (15) or unaffected (16). Contrasting results were reported by Kloetzel and coworkers who showed that transfection of Lmp2 and/or Lmp7 in both T2 and the mouse B8 fibroblasts correlated with decreased chymotrypsin-like and peptidylglutamyl hydrolyzing activities (43, 44, 45). The discrepancy may be explained by differences in the protocols used for proteasome purification, which could result in variable degrees of contamination by other cellular proteases. The significance of these findings for the production of antigenic peptides is also unclear. While the presence of Lmp2 and Lmp7 appears to be important for the generation of certain immunogenic peptides, such as the immunodominant epitope from the murine CMV immediate early Ag pp89 (41, 44), these ß subunits are clearly not essential for the production of other CTL epitopes derived from the influenza nucleoprotein and matrix protein (46, 47, 48).

Degradation experiments performed with the fluorogenic substrates SucLLVY-MCA and BocLRR-MCA (Fig. 4Go) and the E4416–424Y analogue (Fig. 6Go) indicate that proteasomes derived from both BL cells and the A1 transfectant have a different cleavage specificity compared with proteasomes derived from LCLs. The low expression of Lmp2 and Lmp7 correlated with less efficient cleavage of hydrophobic and basic fluorogenic substrates (cf Fig. 1Go and Fig. 4Go, Fig. 5GoA and 5B). This was not an artifact due to contamination by other proteases, such as aminopeptidases, since the cleavage of fluorogenic substrates was not influenced by a wide panel of protease inhibitors. The E4416–424Y analogue was cleaved by proteasomes from BLs but was completely resistant to cleavage by proteasomes from LCLs (Fig. 6Go). Treatment of BL cells with IFN-{gamma} resulted in up-regulation of Lmp2 and Lmp7, down-regulation of {delta}, and enhanced chymotrypsin- and trypsin-like activities against fluorogenic substrates (Fig. 7Go, A–C) but did not affect the cleavage of the E4416–424Y peptide (Fig. 8Go, A and B). This indicates that the enzymatic activity involved in the degradation of E4416–424Y is not directly modulated by Lmp2 and Lmp7. Conceivably, other catalytic subunits may be responsible for cleavage of this substrate. At least five enzymatic activities have been associated with the proteasome including chymotrypsin-like activity, trypsin-like activity, peptidylglutamyl hydrolyzing activity, branch chain amino acid preferring activity, and cleavage between small neutral amino acids (reviewed in 14). The E4416–424Y peptide contains at least three sites for branch chain amino acid preferring activity (P2, P7, and P8) and one site for the peptidylglutamyl hydrolyzing activity (P4). It is not known which ß subunits confer these specificities. The constitutively expressed {delta}, MB1, and MC14 subunits may be involved. If this is the case, our failure to modulate the cleavage specificity by IFN-{gamma} treatment may be due to the relatively long half-life of the proteasome (49), which may result in the persistence of significant levels of these subunits. Alternatively, the proteasome specificity detected in both BL cells and the A1 subline may be due to differential expression of regulatory components of the proteasome complex. Recent reports have focused the attention on the PA28{alpha} subunit that is known to associate with the 20S core (50, 51). Addition of PA28 to purified 20S proteasomes was shown to enhance the production of immunogenic nonamer peptides from longer precursors (50). This correlated with a more efficient presentation of the immunodominant epitope from murine CMV pp89 in mouse fibroblasts transfected with human or mouse PA28 (51). So far, we have been unable to test the expression of PA28 in BL cells due to a lack of specific reagents, and we cannot exclude its involvement. However, PA28 was shown to be induced by IFN-{gamma} treatment (52), and it is therefore unlikely that differences in the expression of this regulatory subunit would be sufficient to explain the degradation of E4416–424Y substrate by proteasomes from untreated and IFN-{gamma}-treated BL cells. Finally, although we have clearly shown that the cleavage of the E4416–424Y peptide was inhibited by the proteasome-specific inhibitor lactacystin and by the peptide aldehyde z-LLL (Fig. 6Go and data not shown), we cannot formally exclude the possibility that other as yet unidentified proteases may be sensitive to these inhibitors and may therefore be responsible for the enzymatic activity observed in BL cells. It is noteworthy that higher concentrations of lactacystin were required to inhibit the degradation of the synthetic peptide compared with the fluorogenic substrates (cf Fig. 3GoB and Fig. 6Go). This is likely due to the different enzymatic activities involved in the cleavage, since lactacystin was shown to inhibit chymotrypsin-like, trypsin-like, and peptidylglutamyl hydrolyzing activities with different kinetics (53).

Taken together, our results strongly indicate that the different peptidase activities of the BL proteasomes may result in the production of characteristic sets of peptides that are different from those produced in other cell types. We have previously shown that BL cells cannot present the E4416–424 epitope even after full reconstitution of TAP function (23). Our present observations may offer an explanation for this phenomenon, since degradation of this peptide may prevent its accumulation in the cytosol. While the significance of this finding for the immunoescape of BL is unclear because the tumor does not express viral Ags that are recognized by EBV-specific CTLs, differences in proteasome cleavage specificity could provide an efficient route of immunoescape in other EBV-associated malignancies, such as nasopharyngeal carcinoma and Hodgkin’s disease, which express at least some of the highly immunogenic viral Ags serving as targets for the efficient rejection of LCL cells (reviewed in 54 . Indeed, down-regulation of the proteasome ß subunits Lmp2 and Lmp7 has been shown to favor immunoescape in other tumor models such as renal cell carcinoma and small lung carcinoma (55, 56).

We have shown that differences in proteasome subunit composition and cleavage specificity are associated with the particular stage of B cell activation/differentiation represented by the BL tumor. This finding has interesting implications for understanding the life-long persistence of EBV-infected cells in healthy virus carriers in the face of strong CTL responses. The virus has been detected in circulating "resting" B cells (57) that, similar to BL, express only LMP2A and EBNA1 (58, 59, 60). EBNA1 may not act as a rejection target, due to the presence of an internal Gly-Ala domain that prevents Ag processing (61), whereas several CTL-epitopes have been mapped within the LMP2A sequence (reviewed in 62 . Some of these epitopes are presented in a TAP-independent fashion (63). It is tempting to speculate that these virus-infected resting B cells may not be subject to EBV-specific CTL surveillance because their proteasomes cannot generate the relevant antigenic peptides.


   Acknowledgments
 
We thank Drs. J. Trowsdale and I. Correa for the kind gift of the Lmp2-, Lmp7-, MECL-1-, and {delta}-specific rabbit sera; Dr. K.B. Hendil for the mAb MCP21; and Dr. B. Kempkes for the ER/EB2–5 cell line.


   Footnotes
 
1 This work was supported by grants from the Swedish Cancer Society, the Childrens Cancer Foundation, the Karolinska Institute, Konung Gustaf V:s Jubileumsfond, and the Magnus Bergvalls Stiftelse, Stockholm, Sweden. Back

2 Address correspondence and reprint requests to Dr. Teresa Frisan, MTC Karolinska Institute, Box 280, S-171 77 Stockholm, Sweden. E-mail address: Back

3 Abbreviations used in this paper: ER, endoplasmic reticulum; BL, Burkitt’s lymphoma; EBNA, EBV nuclear Ag; LCL, EBV-transformed lymphoblastoid cell line; Lmp, low molecular mass polypeptide; LMP, latent membrane protein; NEPHGE, nonequilibrium pH gel electrophoresis; FPLC, fast performance liquid chromatography; 2D, two-dimensional. Back

Received for publication August 20, 1997. Accepted for publication December 9, 1997.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Townsend, A., J. Trowsdale. 1993. The transporters associated with antigen presentation. Semin. Cell Biol. 4:53.[Medline]
  2. Rock, K. L., C. Gramm, L. Rothstein, K. Clark, R. Stein, L. Dick, D. Hwang, A. L. Goldberg. 1994. Inhibitors of the proteasome block the degradation of most cell proteins and the generation of peptides presented on MHC class I molecules. Cell 78:761.[Medline]
  3. Michalek, M. T., E. P. Grant, C. Gramm, A. L. Goldberg, K. L. Rock. 1993. A role for ubiquitin-dependent proteolytic pathway in MHC class I-restricted antigen presentation. Nature 363:552.[Medline]
  4. Lupas, A., A. J. Koster, W. Baumeister. 1993. Structural features of 26S and 20S proteasomes. Enzyme Protein 47:252.[Medline]
  5. Tanaka, K., T. Tamura, T. Yoshimura, A. Ichihara. 1992. Proteasomes: protein and gene structure. New Biol. 4:173.[Medline]
  6. Zwickl, P., A. Grziwa, G. Puhler, B. Dahlmann, F. Lottspeich, W. Baumeister. 1992. Primary structure of the Thermoplasma proteasome and its implications for the structure, function and evolution of the multicatalytic proteinase. Biochemistry 31:964.[Medline]
  7. Seemüller, E., A. Lupas, D. Stock, J. Löwe, R. Huber, W. Baumeister. 1995. Proteasome from Thermoplasma acidophylum: a threonine protease. Science 268:579.[Abstract/Free Full Text]
  8. Glynne, R., S. Powis, S. Beck, A. Kelly, L. Kerr, J. Trowsdale. 1991. A proteasome-related gene between the two ABC transporter loci in the class II region of the human MHC. Nature 353:357.[Medline]
  9. Kelly, A., S. Powis, R. Glynne, E. Radley, S. Beck, J. Trowsdale. 1991. Second proteasome-related gene in the human MHC class II region. Nature 353:667.[Medline]
  10. Monaco, J., H. McDevitt. 1984. H-2-linked low-molecular weight polypeptide antigens assemble into an unusual macromolecular complex. Nature 309:797.[Medline]
  11. Aki, M., N. Shimbara, M. Takashina, K. Akiyama, S. Kagawa, T. Tamura, N. Tanahashi, T. Yoshimura, K. Tanaka, A. Ichihara. 1994. Interferon-{gamma} induces different subunit organizations and functional diversity of proteasomes. J. Biochem. 115:257.[Abstract/Free Full Text]
  12. Groettrup, M., R. Kraft, S. Kostka, S. Standera, R. Stohwasser, P. M. Kloetzel. 1996. A third interferon-{gamma}-induced subunit exchange in the 20S proteasome. Eur. J. Immunol. 26:863.[Medline]
  13. Hisamatsu, H., N. Shimbara, Y. Saito, P. Kristensen, K. B. Hendil, T. Fujiwara, E. Takahashi, N. Tanahashi, T. Tamura, A. Ichihara, K. Tanaka. 1996. Newly identified pair of proteasomal subunits regulated reciprocally by interferon-{gamma}. J. Exp. Med. 183:1807.[Abstract/Free Full Text]
  14. Cardozo, C.. 1993. Catalytic components of the bovine pituitary multicatalytic proteinase complex (proteasome). Enzyme Protein 47:296.[Medline]
  15. Gaczynska, M., K. L. Rock, A. L. Goldberg. 1993. Nature 365:264.[Medline]
  16. Driscoll, J., M. G. Brown, D. Finley, J. J. Monaco. 1993. MHC-linked LMP gene products specifically alter peptidase activities of the proteasome. Nature 365:262.[Medline]
  17. Gregory, C. D., T. Tursz, C. F. Edwards, C. Tetaud, M. Talbot, B. Caillou, A. B. Rickinson, M. Lipinski. 1987. Identification of a subset of normal B cells with a Burkitt’s lymphoma (BL)-like phenotype. J. Immunol. 139:313.[Abstract]
  18. Rickinson, A., R. Murray, J. Brooks, H. Griffin, D. Moss, M. Masucci. 1992. T cell recognition of Epstein-Barr virus associated lymphomas. A. McMichael, and L. Franks, eds. In A New Look at Tumor Immunology Vol. 13:53.-80. CSHL Press, New York.
  19. Rowe, M., D. T. Rowe, C. D. Gregory, L. S. Young, P. J. Farrell, H. Rupani, A. B. Rickinson. 1987. Differences in B cell growth phenotype reflect novel patterns of Epstein-Barr virus latent gene expression in Burkitt’s lymphoma. EMBO J. 6:2743.[Medline]
  20. Rooney, C. M., M. Rowe, L. E. Wallace, A. B. Rickinson. 1985. Epstein-Barr virus-positive Burkitt’s lymphoma cells not recognized by virus-specific T-cell surveillance. Nature 317:629.[Medline]
  21. Khanna, R., S. R. Burrows, V. Argaet, D. J. Moss. 1994. Endoplasmic reticulum signal sequence facilitated transport of peptide epitopes restores immunogenicity of an antigen processing defective tumour cell line. Int. Immunol. 6:639.[Abstract/Free Full Text]
  22. Rowe, M., R. Khanna, C. A. Jacob, V. Argaet, A. Kelly, S. Powis, M. Belich, D. Croom-Carter, S. Lee, S. R. Burrows, J. Trowsdale, D. J. Moss, A. B. Rickinson. 1995. Restoration of endogenous antigen processing in Burkitt’s lymphoma cells by Epstein-Barr virus latent membrane protein-1: coordinate up-regulation of peptide transporters and HLA-class I antigen expression. Eur. J. Immunol. 25:1374.[Medline]
  23. Frisan, T., Q.-J. Zhang, J. Levitskaya, M. Coram, M. G. Kurilla, M. G. Masucci. 1996. Defective presentation of MHC class I-restricted cytotoxic T-cell epitopes in Burkitt’s lymphoma cells. Int. J. Cancer 68:251.[Medline]
  24. Lenoir, G. M., M. Vuillaume, C. Bonnardel. 1985. The use of lymphomatous and lymphoblastoid cell lines in the study of Burkitt’s lymphoma. G. M. Lenoir, and G. T. O’Conor, and C. L. M. Olweny, eds. In Burkitt’s Lymphoma: A Human Cancer Model Vol. 60:309.-318. International Agency for Research on Cancer Scientific Publications, Lyon.
  25. Diabata, M., R. E. Humphreys, K. Takada, T. Sairenji. 1990. Activation of latent EBV via anti-IgG-triggered, second messenger pathways in the Burkitt’s lymphoma cell line Akata. J. Immunol. 144:4788.[Abstract]
  26. Rooney, C. M., C. D. Gregory, M. Rowe, S. Finerty, C. Edwards, H. Rupani, A. B. Rickinson. 1986. Endemic Burkitt’s lymphoma: phenotypic analysis of tumour biopsy cells and of the derived tumour cell lines. J. Natl. Cancer Inst. 77:681.
  27. de Campos-Lima, P. O., V. Levitsky, J. Brooks, S. P. Lee, L. F. Hu, A. B. Rickinson, M. G. Masucci. 1994. T cell responses and virus evolution: loss of HLA A11-restricted CTL epitopes in Epstein-Barr virus isolates from highly A11-positive populations by selective mutation of anchor residues. J. Exp. Med. 179:1297.[Abstract/Free Full Text]
  28. Salter, R. D., P. Cresswell. 1986. Impaired assembly and transport of HLA-A and -B antigens in a mutant TxB cell hybrid. EMBO J. 5:943.[Medline]
  29. Kempkes, B., D. Spitkovsky, P. Jansen-Dürr, J. W. Ellwart, E. Kremmer, H.-J. Delecluse, C. Rottenberg, G. W. Bornkamm, W. Hammerschmidt. 1995. B-cell proliferation and induction of early G1-regulating proteins by Epstein-Barr virus mutants conditional for EBNA2. EMBO J. 14:88.[Medline]
  30. Polack, A., K. Hörtnagel, A. Pajic, B. Christoph, B. Baier, M. Falk, J. Mautner, C. Geltinger, G. W. Bornkamm, B. Kempkes. 1996. c-myc activation renders proliferation of Epstein-Barr virus (EBV)-transformed cells independent of EBV nuclear antigen 2 and latent membrane protein 1. Proc. Natl. Acad. Sci. USA 93:10411.[Abstract/Free Full Text]
  31. Hirs, C., S. Timasheff. 1977. Methods of peptide synthesis. Methods Enzymol. 47:501.[Medline]
  32. Laemmli, U. K.. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680.[Medline]
  33. Glynne, R., L.-A. Kerr, I. Mockridge, S. Beck, A. Kelly, J. Trowsdale. 1993. The major histocompatibility complex-encoded proteasome component LMP7: alternate first exon and post-translational processing. Eur. J. Immunol. 23:860.[Medline]
  34. Mann, K. P., D. Staunton, D. A. Thorley-Lawson. 1985. Epstein-Barr virus-encoded protein found in the plasma membranes of transformed cells. J. Virol. 55:710.[Abstract/Free Full Text]
  35. Hendil, K. B., P. Kristensen, W. Uerkvitz. 1995. Human proteasomes analysed with monoclonal antibodies. Biochem. J. 305:245.
  36. Blum, H., H. Beier, H. J. Gross. 1987. Improved silver staining of plant protein, RNA and DNA in polyacrylamide gels. Electrophoresis 8:93.
  37. Lehninger, A. L., D. L. Nelson, M. M. Cox. 1993. Principles of Biochemistry Worth Publishers, New York.
  38. Heemels, M. T., T. N. Schumacher, K. Wonigeit, H. L. Ploegh. 1993. Peptide translocation by variants of the transporter associated with antigen processing. Science 262:2059.[Abstract/Free Full Text]
  39. Schägger, H., G. von Jagow. 1987. Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for separation of proteins in the range from 1 to 100 kDa. Anal. Biochem. 166:368.[Medline]
  40. Hoffman, L., M. Rechsteiner. 1994. Activation of the multicatalytic protease. J. Biochem. 269:16890.
  41. Boes, B., H. Hengel, T. Ruppert, G. Multhaup, U. H. Koszinowski, P.-M. Kloetzel. 1994. Interferon {gamma} stimulation modulates the proteolytic activity and cleavage site preference of 20S mouse proteasomes. J. Exp. Med. 179:901.[Abstract/Free Full Text]
  42. Brown, M. G., J. Driscoll, J. J. Monaco. 1993. MHC-linked low molecular mass polypeptide subunits define distinct subsets of proteasomes. J. Immunol. 151:1193.[Abstract]
  43. Groettrup, M., T. Ruppert, L. Kuehn, M. Seeger, S. Standera, U. Koszinowski, P.-M. Kloetzel. 1995. The interferon-{gamma}-inducible 11S regulator (PA28) and the LMP2/LMP7 subunits govern the peptide production by the 20S proteasome in vitro. J. Biol. Chem. 270:23808.[Abstract/Free Full Text]
  44. Kuckelkorn, U., S. Frentzel, R. Kraft, S. Kostka, M. Groettrup, P.-M. Kloetzel. 1995. Incorporation of major histocompatibility complex-encoded subunits LMP2 and LMP7 changes the quality of the 20S proteasome polypeptide processing products independent of interferon-{gamma}. Eur. J. Immunol. 25:2605.[Medline]
  45. Stohwasser, R., U. Kuckelkorn, R. Kraft, S. Kostka, P. M. Kloetzel. 1996. 20S proteasome from LMP7 knock out mice reveals altered proteolytic activities and cleavage site preferences. FEBS Lett. 383:109.[Medline]
  46. Driscoll, A. D., M. Androlewicz, E. Hughes, P. Cresswell, T. Spies. 1992. Proteasome subunits encoded in the MHC are not generally required for the processing of peptides bound by MHC class I molecules. Nature 360:171.[Medline]
  47. Momburg, F., V. Ortiz-Navarrete, J. Neefjes, E. Goulmy, Y. van de Wal, H. Spits, S. J. Powis, G. W. Butcher, J. C. Howard, P. Walden, G. J. Hämmerling. 1992. Proteasome subunits encoded by the major histocompatibility complex are not essential for antigen presentation. Nature 360:174.[Medline]
  48. Zhou, X., F. Momburg, T. Liu, U. M. Abdel Montal, M. Jondal, G. J. Hämmerling, H.-G. Ljunggren. 1994. Presentation of viral antigens restricted by H-2Kb, Db or Kd in proteasome subunit LMP2- and LMP7-deficient cells. Eur. J. Immunol. 24:1863.[Medline]
  49. Hendil, K. B.. 1988. The 19S multicatalytic "prosome" proteinase is a constitutive enzyme in HeLa cells. Biochem. Int. 17:471.[Medline]
  50. Dick, P. T., T. Ruppert, M. Groettrup, P. M. Kloetzel, L. Kuehn, U. H. Koszinowski, S. Stevanovic, H. Schild, H.-G. Rammensee. 1996. Coordinated dual cleavages induced by proteasome regulator PA28 lead to dominant MHC ligands. Cell 86:253.[Medline]
  51. Groettrup, M., A. Soza, M. Eggers, L. Kuehn, T. P. Dick, H. Schild, H.-G. Rammensee, U. H. Koszinowski, P. M. Kloetzel. 1996. A role for the proteasome regulator PA28. Nature 381:166.[Medline]
  52. Ahn, J. Y., N. Tanahashi, K. Akiyama, H. Hisamatsu, C. Noda, K. Tanaka, C. H. Chung, N. Shimbara, P. J. Willy, J. D. Mott, C. A. Slaughter, G. N. DeMartino. 1995. Primary structures of two homologue subunits of PA28, a gamma-interferon-inducible protein activator of the 20S proteasome. FEBS Lett. 366:37.[Medline]
  53. Fenteany, G., R. F. Standaert, W. S. Lane, S. Choi, E. J. Corey, S. L. Schreiber. 1995. Inhibition of proteasome activities and subunit-specific amino-terminal threonine modification by lactacystin. Science 268:726.[Abstract/Free Full Text]
  54. de Campos-Lima, P.-O., J. Levitskaya, T. Frisan, M. G. Masucci. 1996. Strategies of immunoescape in Epstein-Barr virus persistence and pathogenesis. Semin. Virol. 7:75.
  55. Restifo, N. P., F. Esquivel, Y. Kawakami, J. W. Yewdell, J. J. Mule, S. A. Rosenberg, J. R. Bennink. 1993. Identification of human cancers deficient in antigen processing. J. Exp. Med. 177:265.[Abstract/Free Full Text]
  56. Selinger, B., A. Höhne, A. Knuth, H. Bernhard, T. Meyer, R. Tampe, F. Momburg, C. Huber. 1995. Analysis of the major histocompatibility complex class I antigen presentation machinery in normal and malignant renal cells: evidence for deficiencies associated with transformation and progression. Cancer Res. 56:1756.[Abstract/Free Full Text]
  57. Miyashita, E. M., B. Yang, K. M. Lam, D. H. Crawford, D. A. Thorley-Lawson. 1995. A novel form of Epstein-Barr virus latency in normal B cells in vivo. Cell 80:593.[Medline]
  58. Qu, L., D. T. Rowe. 1992. Epstein-Barr virus latent gene expression in uncultured peripheral blood lymphocytes. J. Virol. 66:3715.[Abstract/Free Full Text]
  59. Chen, F., J.-Z. Zou, L. Di Renzo, G. Winberg, L.-F. Hu, E. Klein, G. Klein, E. Ernberg. 1995. A subpopulation of normal B cells latently infected with Epstein-Barr virus resembles Burkitt lymphoma cells in expressing EBNA-1 but not EBNA-2 or LMP1. J. Virol. 69:3752.[Abstract]
  60. Tierney, R. J., N. Steven, L. S. Young, A. B. Rickinson. 1994. Epstein-Barr virus latency in blood mononuclear cells: analysis of viral gene transcription during primary infection and in the carrier state. J. Virol. 68:7374.[Abstract/Free Full Text]
  61. Levitskaya, J., M. Coram, V. Levitsky, S. Imreh, P. M. Steigerwald-Mullen, G. Klein, M. G. Kurilla, M. G. Masucci. 1995. Inhibition of antigen processing by the internal repeat region of the Epstein-Barr virus nuclear antigen-1. Nature 375:685.[Medline]
  62. Masucci, M. G., I. Ernberg. 1994. Epstein-Barr virus adaptation to a life within the immune system. Trends Microbiol. 2:125.[Medline]
  63. Lee, S. P., W. A. Thomas, N. W. Blake, A. B. Rickinson. 1996. Transporter (TAP)-independent processing of a multiple membrane-spanning protein, the Epstein-Barr virus latent membrane protein 2. Eur. J. Immunol. 26:1875.[Medline]



This article has been cited by other articles:


Home page
 J. Immunol.Home page
S. M. Walton, M. Gerlinger, O. de la Rosa, N. Nuber, A. Knights, A. Gati, M. Laumer, L. Strauss, C. Exner, N. Schafer, et al.
Spontaneous CD8 T Cell Responses against the Melanocyte Differentiation Antigen RAB38/NY-MEL-1 in Melanoma Patients
J. Immunol., December 1, 2006; 177(11): 8212 - 8218.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
M. J. Palmowski, U. Gileadi, M. Salio, A. Gallimore, M. Millrain, E. James, C. Addey, D. Scott, J. Dyson, E. Simpson, et al.
Role of Immunoproteasomes in Cross-Presentation
J. Immunol., July 15, 2006; 177(2): 983 - 990.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
R. Gavioli, E. Gallerani, C. Fortini, M. Fabris, A. Bottoni, A. Canella, A. Bonaccorsi, M. Marastoni, F. Micheletti, A. Cafaro, et al.
HIV-1 Tat Protein Modulates the Generation of Cytotoxic T Cell Epitopes by Modifying Proteasome Composition and Enzymatic Activity
J. Immunol., September 15, 2004; 173(6): 3838 - 3843.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
B. Kessler, X. Hong, J. Petrovic, A. Borodovsky, N. P. Dantuma, M. Bogyo, H. S. Overkleeft, H. Ploegh, and R. Glas
Pathways Accessory to Proteasomal Proteolysis Are Less Efficient in Major Histocompatibility Complex Class I Antigen Production
J. Biol. Chem., March 14, 2003; 278(12): 10013 - 10021.
[Abstract] [Full Text] [PDF]

Home page