Loading Proteasome Activity and MHC Class I Peptide Allelic Differences in the Relationship Between

Allelic Differences in the Relationship Between Proteasome Activity and MHC Class I Peptide Loading

This information is current as Adam M. Benham, Monique Grommé and Jacques Neefjes of September 24, 2021. J Immunol 1998; 161:83-89; ; http://www.jimmunol.org/content/161/1/83 Downloaded from References This article cites 48 articles, 24 of which you can access for free at: http://www.jimmunol.org/content/161/1/83.full#ref-list-1

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 1998 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Allelic Differences in the Relationship Between Proteasome Activity and MHC Class I Peptide Loading1

Adam M. Benham, Monique Gromme´, and Jacques Neefjes2

MHC class I molecules are cell surface glycoproteins that play a pivotal role in the response to intracellular pathogens. The loading of MHC class I molecules with antigenic substrates takes place in the endoplasmic reticulum. This requires a functional TAP transporter, which translocates peptides into the endoplasmic reticulum from the cytosol. The generation of antigenic peptides from polypeptide precursors is thought to be mediated in the cytosol by the proteasome. Previously, we have demonstrated that inhibiting the proteasome with the speciﬁc covalent inhibitor lactacystin results in a direct reduction of peptide-loaded MHC class I molecules. This indicates that the proteasome is the limiting step in the MHC class I pathway. In this study we use isoelectric focusing to demonstrate that two related MHC class I alleles, HLA-A3 and HLA-A11, as well as HLA-B35 do not follow this behavior. In contrast to other class I alleles expressed by the same cells, these alleles are loaded with peptides and mature normally Downloaded from when proteasome activity is severely inhibited. Our observations highlight a new level of diversity in the MHC class I system and indicate that there are allele-speciﬁc differences in the linkage between proteasome activity and MHC class I peptide loading. The Journal of Immunology, 1998, 161: 83–89.

efense against intracellular pathogens and tumor cells is studies performed with fluorogenic peptide substrates. These in- ϩ mediated by CD8 T cells. These effector cells specif- clude a chymotrypsin-like activity and a trypsin-like activity, http://www.jimmunol.org/ D ically recognize foreign peptides in the context of self which catalyze scission of the peptide bond on the C-terminal side MHC class I molecules expressed at the plasma membrane, thus of hydrophobic and basic amino acids, respectively. An indepen- orchestrating the specific lysis of the infected or dysfunctional cell dent acid-like enzyme activity has been postulated to be involved (1). Antigenic peptides have to be generated from polypeptide pre- in cleavage at the N-terminus of the substrate, with the products of cursors by cytosolic protease activity (2, 3) before their transport proteasome digestion being around nine amino acids in length from the cytosol into the endoplasmic reticulum (ER)3 by the (11). In the assembled eukaryotic proteasome, only three of the ATP-dependent peptide transporter TAP (4). TAP preferentially seven types of ␤ subunits are catalytically active. The generation translocates peptides of between 8 and 14 amino acids in length of these active sites takes place during proteasome assembly by by guest on September 24, 2021 with some sequence specificity (5, 6). Within the ER, antigenic cleavage of an N-terminal pro-sequence that exposes the catalytic peptides associate with an MHC class I heterodimer (a heavy chain threonine (12, 13). In mammals, these three catalytic subunits, ␤1 ␤ and the light chain 2m) to form a functional trimeric complex. (Y/␦), ␤2 (Z), and ␤5 (X/MB1), are co-ordinately replaced upon This exits the ER and traverses the Golgi apparatus where it ac- IFN-␥ stimulation by the active ␤ subunits, LMP2, MECL1, and quires sialic acid modifications before arrival at the plasma LMP7, respectively (14, 15). Examination of the Saccharomyces membrane. cerevisae proteasome crystal structure suggests that the incorpo- A wealth of experimental data has implicated the proteasome in ration of LMP2 into the particle should enhance the generation of the generation of antigenic peptides (7). The proteasome is a mul- peptides with C-terminal hydrophobic and basic residues at posi- ticatalytic particle consisting of structural ␣ subunits and catalytic tion 9 (11, 16, 17). This makes them more suitable for binding to ␤ ␣ ␤ ␤ ␣ subunits arranged as an 7 7 7 7 cylinder (8). The proteasome MHC class I molecules that accommodate such residues in their F moves within the cell by diffusion (9) and is a member of the pockets. Consistent with this, LMP2- and LMP7-deficient mice Ntn-hydrolase family of enzymes, since active ␤ subunits posses a and cell lines have been claimed to express diminished amounts of catalytic N-terminal threonine residue (10). Up to five types of MHC class I molecules at the cell surface, although the phenotypes catalytic activities have been ascribed to the proteasome based on are rather mild (18, 19). Functional evidence for a role of the proteasome in the class I Ag presentation pathway also comes from studies using broad Division Cellular Biochemistry, Netherlands Cancer Institute, Amsterdam, The Neth- specificity inhibitors, which block the presentation of endogenous erlands antigens to CTL (20, 21). More recently, a Streptomyces metabo- Received for publication December 2, 1997. Accepted for publication February 27, lite called lactacystin and its ␤-lactone derivative have been shown 1998. to bind specifically and covalently to the catalytic subunits of The costs of publication of this article were defrayed in part by the payment of page mammalian proteasomes (22–24). Irreversible inhibition of pro- charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. teasomes using lactacystin specifically abolishes the presentation 1 This work was supported by European Community Fellowship EBCHBGCT930356 of a range of viral proteins to CTL (25), although this may not be (to A.B.) and Netherlands Organization for Scientific Research Grant 901-09-027 (to the case for all antigenic peptides (26). M.G.). In vivo, the proteasome interacts with modulatory protein com- 2 Address correspondence and reprint requests to Dr. Jacques Neefjes, Division Cel- plexes. Included among these is the 19S cap, which is involved in lular Biochemistry, Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Am- sterdam, The Netherlands. E-mail address: [email protected] the targeting of ubiquitinated proteins to the proteasome (27), and ␥ 3 Abbreviations used in this paper: ER, endoplasmic reticulum; PSI, Cbz-Ile-Glu(O- the IFN- -inducible activator PA28 (28). PA28 exists as a hex- t-Bu)-Ala-Leu; 1D-IEF, one-dimensional isoelectric focusing. amer of ␣ and ␤ subunits that binds at the ends of the proteasome

cylinder (29). Overexpression of PA28␣ has been shown to en- portions and assayed against 100 ␮M of the peptide substrates Suc-Leu- hance the generation of some Ag for presentation to CTL (30) and Leu-Val-Tyr-AMC and z-Ala-Ala-Arg-AMC (Novabiochem, Laütelfin- in vitro, PA28 alters the pattern of peptide products generated by gen, Switzerland, and Bachem, Bubendorf, Switzerland) in a total volume of 200 ␮l of hypotonic buffer at 37°C. These substrates give an accurate purified 20S proteasome digests (31). However, how PA28 func- determination of the chymotrypsin-like and the trypsin-like activities of the tions in vivo with respect to the 26S proteasome complex in Ag proteasome, respectively. Aliquots of 10 ␮l were quenched in duplicate presentation is unresolved. into 1 ml of ethanol after 8 h, and the fluorescence of the free AMC ␭ ϭ ␭ ϭ Previously, we used the proteasome-specific inhibitor lactacys- ( excitation 370 nm, emission 460 nm) was measured using a spec- tin to demonstrate that the trypsin-like activity of the proteasome trofluorometer (Perkin-Elmer, Den Bosch, The Netherlands). Incubation buffer plus peptides, beads, or Ab alone gave negligible fluorescence. Chy- was closely correlated with the peptide loading of MHC class I motrypsin and trypsin digestion of peptides was used as a positive control. molecules in four different cell types (32). Both the trypsin-like Proteasome activity was calculated by subtracting the mean W6/32 (con- and the chymotrypsin-like activities of the proteasome became re- trol) values from the mean MCP21 values at each concentration and setting ␮ lated to MHC class I stability after IFN-␥ stimulation. These re- the figure obtained at 0 M lactacystin to 100% for each substrate. Control values were always Ͻ10% of specific values. sults indicated that in cells optimized for Ag presentation, the proteasome is the limiting factor in the MHC class I Ag presentation pathway. In this report we show that while this assertion holds for Biosynthetic labeling and MHC class I immunoprecipitations the majority of MHC class I alleles, it is not universal. HLA-A3, MHC class I stability assays. Approximately 2.5 ϫ 106 cells/immuno- HLA-A11, and HLA-B35 do not conform to this model and are precipitation were incubated for2hinthepresence or the absence of resistant to the effects of proteasome inhibitors at concentrations proteasome inhibitor. Cells were then starved for1hincysteine/methio- Downloaded from that inhibit 70 to 80% of the trypsin-like and chymotrypsin-like nine-deficient RPMI medium and 10% FCS (also in the presence or the activities. This is also observed across different cell lines. These absence of inhibitor) and metabolically labeled with 125 ␮Ci [35S]cysteine/ results imply that while peptide generation by the proteasome is methionine (Amersham) per sample for 20 min at 37°C. Aliquots were quenched in 10 ml of cold PBS and lysed in lysis buffer containing 50 mM limiting for most MHC class I alleles, the peptide loading of other Tris-HCl (pH 7.5), 5 mM MgCl2, 150 mM NaCl, and 1% (w/v) Nonidet alleles is free from this limitation. The implications of these find- P-40. Nuclei were removed by centrifugation, and the lysates were pre- ings will be discussed. cleared overnight on protein A-Sepharose beads. Aliquots of 5 ␮l were taken from the lysates and TCA precipitated to monitor the incorporation http://www.jimmunol.org/ of [35S]cysteine/methionine into proteins. Lysates from equal amounts of Materials and Methods TCA-precipitable radioactivity were split into two equal halves. One ali- Chemicals quot was incubated at 37°C for 2 h, and the other aliquot was kept at 4°C. All other steps were performed at 4°C. The lysates were precleared for 1 h The proteasome-specific inhibitor lactacystin (22) was obtained from E. J. with normal mouse serum on protein A-Sepharose beads, and MHC class Corey, Harvard University (Boston, MA), and was stored as a powder or I molecules were subsequently immunoprecipitated using the conforma- as a 10-mM stock solution in sterile water at 4°C. The proteasome inhibitor tion-specific mAb W6/32. Pellets were washed four times in lysis buffer PSI (Cbz-Ile-Glu(O-t-Bu)-Ala-Leu; Calbiochem, La Jolla, CA) and the before analysis by isoelectric focusing (1D-IEF). Quantitation of gels was proteasome inhibitor z-L VS (33) (a gift from Prof. H. Ploegh, Harvard 3 performed using a Fuji X-OMAT PhosphorImager (Fuji, Tokyo, Japan) University) were stored as 10-mM stock solutions at Ϫ20°C in DMSO. equipped with TINA software. The amount of stable MHC class I mole- Human rIFN-␥ was obtained from Boehringer Ingelheim (Ingelheim, Ger- by guest on September 24, 2021 cules retrieved in the absence of proteasome inhibitors was set at 100% so many) and was added to HeLa cells at a concentration of 100 U/ml for 72 h that the relative percent stability of each MHC class I molecule could be or 200 U/ml for 24 h where relevant. Other chemicals were purchased from compared. standard suppliers. Thermoinstability of HLA-A3 and -A11 during early biosynthesis. AF 6 6 Cell lines cells (10 ϫ 10 cells/sample) or HeLa cells (2 ϫ 10 cells/sample) were starved in cysteine/methionine-deficient medium, labeled in a small vol- The human cell lines used in this study and their HLA types are as follows. ume (AF cells in 200 ␮l/dish, HeLa cells in 1 ml/dish) with 250 ␮Ci of AF cells (HLA-A1, -A11, -B35, and -B51) were provided by Dr. R. [35S]methionine/cysteine, and chased for various times. Cells were lysed in Khanna (Queensland Institute of Medical Research, Brisbane, Australia). 1 ml of 1% Nonidet P-40. Nuclei were removed from the lysates by cen- OSH cells (HLA-A2, -A11, -B27, and -B35) were donated by Dr. F. Claas trifugation, and the lysates were split into two equal portions. One-half was (AZL, Leiden, The Netherlands). HRC-5 cells (HLA-A11, -B7, and -B18) incubated at 37°C for 30 min, whereas the other half was maintained at were obtained from the ECACC (Porton Down, U.K.). All these B cell 4°C. MHC class I molecules were immunoprecipitated from the lysates lines were cultured in RPMI supplemented with 8% FCS. The cervical with W6/32 and analyzed by 1D-IEF. carcinoma cell line HeLa (obtained from the American Type Culture Col- Pulse-chase analysis of MHC class I molecules. Biosynthetic labeling lection, Rockville, MD) was believed to express HLA-A3, -A28, and -B35, was performed as described above, except that after the radioactive pulse, but was found to be HLA-B75 positive and HLA-B35 negative and to cells were chased (in the continuous presence of proteasome inhibitors express the HLA-A68 subtype of HLA-28 by DNA typing of the HeLa where relevant) with fresh medium containing 1 mM cysteine and 1 mM cells used in our experiments. This was confirmed by isoelectric focusing. methionine. At the given chase points, equal amounts of cells were HeLa was maintained in DMEM supplemented with 8% FCS. quenched in 10 ml of ice-cold PBS, pelleted, and lysed in lysis buffer. The lysates were precleared for 2 h with normal mouse serum on protein A- Antibodies coupled Sepharose beads and then subjected to immunoprecipitation with The conformation-specific mAb W6/32 recognizes correctly folded MHC W6/32 as before. After washing, the immunoprecipitates were analyzed by class I complexes (34). The proteasome Abs used were MCP21 (a gift from 1D-IEF. K. Hendil, August Krogh Institute, Copenhagen, Denmark), which detects the HC3 (␣2) subunit (35) and IB5 (Organon Teknika, Belgium), which recognizes the iota (␣1) subunit (36). Western blotting Proteasome activity assays Material from the cell lysates used in the proteasome activity assays was monitored to check for equal amounts of proteasomes. Ten micrograms of Proteasome activity assays were performed essentially as described previ- protein was dissolved in reducing loading buffer and subjected to 10% ously (32). Cells cultured in the presence of given proteasome inhibitor SDS-PAGE. Proteins were transferred onto nitrocellulose filters at 150 mA concentrations were lysed in a hypotonic buffer (10 mM triethanolamine, for 1.25 h. After transient staining with 0.4% (w/v) Ponceaux S to verify 10 mM acetic acid, 1 mM EDTA, and 0.25 M sucrose, pH 7.4), disrupted equal loading and correct transfer, the filters were blocked with 1% (w/v) using an EMBL cell cracker, and subsequently kept at 4°C. Equal amounts milk powder before probing with a 1/500 dilution of the proteasome-spe- of cytosolic proteins (ϳ300 ␮g) were used to immunoprecipitate protea- cific Ab IB5. After stringent washing in 150 mM NaCl, 0.05% Tween-20, somes after extensive preclearance. Specific immunoprecipitations were and 10 mM Tris, pH 8.0, secondary rabbit anti-murine peroxidase-coupled performed for 1.5 h using either MCP21 (anti-proteasome) or W6/32 as a Abs were added and detected using an ECL kit (Amersham, Arlington negative control. After washing five times, the pellets were split into equal Heights, IL). The Journal of Immunology 85

Results The peptide loading of HLA-A3 is not limited by proteasome activity In our previous work we analyzed cell lines that had been stimulated with or without IFN-␥ and treated in the presence or the absence of the speciﬁc proteasome inhibitor lactacystin. We observed that the stability of all alleles analyzed decreased in a titratable manner with increasing concentrations of lactacystin and that this correlated with a decrease in proteasome activity assayed under the same conditions (32). However, all the MHC class I alleles that were examined had a preference for a hydrophobic amino acid at position 9 in the antigenic peptide. We were therefore interested to see whether MHC class I alleles capable of ac- commodating a basic amino acid in this position also followed this pattern, i.e., whether their peptide loading was related to the trypsin-like activity of the proteasome. Thus, HeLa cells that had been stimulated with IFN-␥ were treated in culture with increasing con-

centrations of the proteasome inhibitor lactacystin. Downloaded from The cells were metabolically labeled for 20 min, lysed, and split into two equal portions, one of which was incubated at 37°C and the other of which was kept on ice. The conformation-speciﬁc mAb W6/32 was then used to solely immunoprecipitate those 37°C-resistant molecules that had acquired stably bound peptides.

Temperature stability as a result of peptide binding is a well-doc- http://www.jimmunol.org/ umented and sensitive assay for antigenic peptide loading of MHC class I molecules (37). The peptide-loaded MHC class I molecules were analyzed by 1D-IEF (Fig. 1A), which resolves different class I alleles according to their isoelectric point (38). Although the stabilities of HLA-A68 and HLA-B75 are clearly reduced with increasing concentrations of lactacystin, HLA-A3 remains stable FIGURE 1. MHC class I stability and proteasome activity in HeLa ␮ even up to 10 M lactacystin. The stability and peptide loading of cells. A, Stability of MHC class I molecules. HeLa cells were cultured with HLA-A3 are also unaffected in HeLa cells that have not been stim- 100 U/ml recombinant human IFN-␥ for 72 h before (and during) incuba- ulated with IFN-␥ (Fig. 1B) and in HeLa cells that have been tion for 3 h with 0, 2, 5, 8, or 10 ␮M lactacystin. MHC class I molecules by guest on September 24, 2021

treated with another proteasome inhibitor, z-L3VS (Fig. 1C). This were immunoprecipitated from 4 and 37°C lysates of metabolically labeled compound also covalently modifies the active sites of proteasome cells and analyzed by 1D-IEF. The positions of HLA-A68, HLA-B75, ␤ ␤ subunits and specifically inhibits their activity (33), producing HLA-A3, and 2m are indicated. HLA-A68 molecules become visibly heat results similar to those obtained with lactacystin (A. M. Benham, labile at 2 ␮M lactacystin, whereas HLA-A3 is lactacystin resistant. B, The ␥ M. Gromme´, and J. Neefjes, unpublished observations). experiment in A was repeated, but in the absence of IFN- . The complete stability of HLA-A3 under these conditions is indicated. C, The experiment To determine the amount of proteasome activity present in HeLa depicted in B was repeated, except that cells were incubated in the presence cells treated with lactacystin, the peptidase activity of isolated pro- ␮ of 0, 0.5, 1, 2, or 5 M z-L3VS rather than lactacystin. Again, HLA-A3 is teasomes was determined using the fluorogenic substrates z-Ala- stable under these conditions. D, MHC class I stability and proteasome Ala-Arg-AMC (trypsin-like activity) and Suc-Leu-Leu-Val-Tyr- activity. The stabilities of HLA-A3 (f) and HLA-A68 () were quanti- AMC (chymotrypsin-like activity; Fig. 1D). Western blotting of tated by exposing the gel depicted in A to a PhosphorImager. HLA-B75 samples of the cell lysates used in this experiment verifies that could not be accurately quantitated. Proteasomes were isolated from IFN- equal quantities of proteasomes were analyzed in the activity assay ␥-stimulated HeLa cells that had been cultured in the presence of 0, 2, 5, (Fig. 1D, inset). Proteasomes isolated from cells treated with the 8, or 10 ␮M lactacystin for 3 h and assayed against 100 ␮M of the flu- covalent inhibitor lactacystin show irreversible and titratable inhi- orogenic peptide substrates: z-Ala-Ala-Arg-AMC (ⅷ; trypsin-like activity) ϩ bition. As we have previously observed, the chymotrypsin-like ac- and Suc-Leu-Leu-Val-Tyr-AMC ( ; chymotrypsin-like activity). The fluorescence values obtained for each substrate were calculated as described, tivity of the proteasome is more readily inhibited than the trypsin- and bars represent 95% confidence intervals. The chymotrypsin-like activ- like activity. Next, we compared the inhibition of the proteasome ity of the proteasome is more readily inhibited than the trypsin-like activity. with its effect on MHC class I peptide loading by quantitating the HLA-A3 is not altered by the decline in proteasome activity, whereas the temperature-dependent destabilization of class I molecules in the stability of HLA-A68 declines with the trypsin-like activity of the protea- presence of lactacystin (Fig. 1D). Quantitation of HLA-A3 stabil- some. The inset represents equal volumes of cell lysate from each protea- ity confirms that HLA-A3 is stable even when 70 to 80% of the some assay point that have been Western blotted with the proteasome- total proteasome activity is inhibited. This contrasts with HLA- specific mAb IB5, directed against the ␣1 (iota) subunit. Equal amounts of A68, expressed in the same cells, for which peptide loading is proteasomes are assayed at each concentration of lactacystin. directly related to proteasome activity, as noted previously (32).

The HLA-A3 superfamily member HLA-A11 is also peptide loaded during proteasome inhibition -A11, -A31, -A33, and -A68 constitutes the A3-like supertype, and HLA-A3 is one of only five alleles described to date with a peptide they all contain negatively charged acidic residues in the hyper- binding motif that can accommodate the basic residues arginine or variable F pocket of the binding groove to facilitate the loading of lysine at position 9 of the antigenic peptide. This set of HLA-A3, a C-terminal basic peptide (39). 86 CLASS I ALLELES AND PROTEASOME ACTIVITY Downloaded from http://www.jimmunol.org/ FIGURE 2. The peptide loading of HLA-A11 in AF cells. A, AF cells FIGURE 3. Thermolability of MHC class I molecules in AF and HeLa ␮ were incubated for 3 h with 0, 0.5, 1, 2, 5, or 10 M z-L3VS. After cells during early biosynthesis. A, AF cells were metabolically labeled (in biosynthetic labeling for 20 min, MHC class I molecules were immuno- the absence of any inhibitors) for 2 min and chased for 0, 1, 3, 5, or 10 min. precipitated from 4 and 37°C lysates and analyzed by 1D-IEF as described MHC class I molecules were immunoprecipitated using W6/32 and ana- in Figure 1. The positions of HLA-A1, HLA-B35, HLA-B51, HLA-A11, lyzed by 1D-IEF after the lysates had been incubated at either 4 or 37°C. ␤ and 2m are indicated. HLA-A11 is resistant to z-L3VS, whereas the other At early chase times, notably 1 and 3 min, all alleles, including HLA-A11, alleles in the same cell line are not. B, Two experiments, including the gel are temperature sensitive. At later chase times, all molecules contain bound in A, were quantitated using a PhosphorImager and show the decrease in peptide and are thermostable. B, HeLa cells were pretreated for 24 h with stability for HLA-A1 () compared with that of HLA-A11 (f) and HLA- 200 U/ml recombinant human IFN-␥ before pulse-chase analysis as de- by guest on September 24, 2021 B35 (Œ). Proteasomes were isolated from AF cells that had been cultured scribed in A. At early chase times, such as 3 min, both HLA-A68 and ␮ at the same concentrations of z-L3VS and assayed against 100 M of either HLA-A3 are temperature sensitive and only become stable after longer z-Ala-Ala-Arg-AMC (ⅷ) or Suc-Leu-Leu-Val-Tyr-AMC (ϩ). The fluo- chase times when appropriate peptides have been acquired. The relatively rescence values obtained for each substrate were calculated as described, weak labeling of HeLa MHC class I molecules with short pulses precludes and bars represent 95% confidence intervals. As with HeLa, the chymot- the visualization of HLA-B75 and the 0 min chase point. rypsin-like activity of the proteasome is more readily inhibited than the trypsin-like activity.

HLA-B35 is also largely resistant to z-L3VS. Experiments using lactacystin gave similar results (not shown). To determine whether any other members of the HLA-A3 fam- It is theoretically possible that HLA-A3 and HLA-A11, unlike ily were also resistant to proteasome inhibition, we analyzed HLA- other MHC class I alleles, are thermostable per se in the absence A11 expressed in the B cell line AF. AF cells were treated in the of peptide and therefore do not dissociate under the conditions presence of increasing concentrations of the proteasome inhibitor used in these assays. Our previous experiments showed that early ␤ z-L3VS and metabolically labeled for 20 min. MHC class I mol- during MHC class I biosynthesis, heavy chain/ 2m heterodimers ecules were immunoprecipitated from cell lysates and analyzed by form that have not yet become peptide loaded and thus pass 1D-IEF as before. Figure 2A shows that HLA-A1 and -B51 be- through a temperature-sensitive stage (40). We made use of these come titratably unstable upon proteasome inhibition. However, un- observations to verify that HLA-A3 and HLA-A11 fold normally der exactly the same conditions and within the same cells, HLA- and are transiently thermolabile before peptide binding. Hence, a

A11 is completely unaffected by z-L3VS. The mean stability of short pulse-chase experiment was performed using both AF cells HLA-A11 was compared with those of HLA-A1 and -B35 and was (Fig. 3A) and HeLa cells (Fig. 3B). Cells were metabolically la- determined in two separate experiments by analyzing the IEF gels beled for 2 min and then chased from 0 to 10 min after the pulse using a PhosphorImager (Fig. 2B). This analysis conﬁrms that before detergent lysis. Thermostability was assayed by incubating HLA-A11 remains stable while HLA-A1 does not. Note that HLA- one-half of the lysate at 4°C and the other half at 37°C before

B35 is also quite stable in the presence of z-L3VS. immunoprecipitation with W6/32 and analysis by 1D-IEF. To elucidate the extent of proteasome inhibition in this cell line, Figure 3 reveals that during the early stages of biosynthesis, at

proteasomes were isolated from AF cells treated with z-L3VS. 1 min for AF (Fig. 3A) and 3 min for HeLa (Fig. 3B), a large Both the chymotrypsin-like and the trypsin-like activities of the majority of MHC class I molecules, including HLA-A3 and HLA-

proteasome are titratably inhibited by z-L3VS in AF cells. Figure A11, are temperature sensitive and not yet peptide loaded. Con- 2B clearly indicates that while inhibition of the trypsin-like activity versely, after 10-min chase, all the MHC class I alleles become of the proteasome is mirrored by inhibition of the loading of HLA- stably peptide loaded and no longer dissociate at 37°C. Thus, like A1, no such relationship exists for HLA-A11. Peptide loading of the other MHC class I molecules that were previously tested (40), The Journal of Immunology 87

FIGURE 4. Resistance of HLA-A11 to three classes of proteasome inhibitor. AF cells were incubated for3hinthepresence of 10 and 50 ␮M ␮ lactacystin, 10 M z-L3VS, and PSI or with DMSO only and metabolically labeled as described. MHC class I molecules were immunoprecipitated using W6/32 and analyzed by 1D-IEF after the lysates had been incubated at either 4 or 37°C. HLA-A11 and -B35 are resistant to the effects of FIGURE 5. Peptide loading of HLA-A11 in OSH and HRC-5 cells. proteasome inhibitors, whereas HLA-A1 and -B51 are not. OSH and HRC-5 cells were incubated for3hinthepresence of 0, 1, 2, 5, or 10 ␮M lactacystin. Proteasomes were isolated from these cells and as- Downloaded from sayed against 100 ␮M of either z-Ala-Ala-Arg-AMC (ⅷ) or Suc-Leu-Leu- Val-Tyr-AMC (ϩ) for OSH cells (solid lines) or HRC-5 cells (dashed HLA-A3 and -A11 are transiently thermolabile early during lines). The fluorescence values obtained for each substrate were calculated biosynthesis. as described. The chymotrypsin-like activity of the proteasome is more To show that the allele-specific effects on peptide loading seen readily inhibited than the trypsin-like activity for both cell lines. The sta- to date were indeed due to the specific effects on proteasome ac- bility of HLA-A11 class I molecules from metabolically labeled cells was tivity, AF cells were cultured in the presence of three different analyzed as described and was quantitated using a PhosphorImager (f). http://www.jimmunol.org/

proteasome inhibitors. z-L3VS is a covalent peptide analogue, PSI HLA-A11 in OSH cells (solid line) and HRC-5 cells (dashed line) are is a noncovalent peptide analogue (41), and lactacystin is a lac- resistant to proteasome inhibition. tone-based metabolite. AF cells were cultured in the presence of each of these inhibitors before immunoprecipitation of peptide- each time point for lysis and immunoprecipitation of their MHC filled class I molecules with W6/32. Figure 4 demonstrates that class I molecules. 1D-IEF analysis (Fig. 6) revealed that the mat- 10-␮M concentrations of all three inhibitors severely reduce the uration of HLA-A1 was compromised by proteasome inhibition, as peptide loading of HLA-A1 and -B51 and have a partial effect on evidenced by the slower disappearance of the immature band (Fig. HLA-B35, but have little effect on the loading of HLA-A11. The 6, species 0). HLA-A11 matured at a similar rate in the presence by guest on September 24, 2021 least specific proteasome inhibitor, PSI, reduces the amount of and the absence of z-L VS (Fig. 6, species 1 and 2). Similar results peptide-loaded HLA-A11 by approximately 10 to 15%. Concen- 3 were obtained when the experiment was repeated using 10 ␮M trations of lactacystin up to 50 ␮M also have little effect on the lactacystin (not shown). Thus, HLA-A11 is efficiently loaded with stability of HLA-A11. To test whether the effects of proteasome inhibitors on HLA- A11 were restricted to a single cell line, two other cell lines were subjected to the same experimental procedures detailed above. OSH cells (expressing HLA-A2, -A11, -B27, and -B35) and HRC-5 cells (expressing HLA-A2, -A11, -B35, and -B40) were both tested for proteasome activity and stability of their MHC class I molecules after treatment with lactacystin. As summarized in Figure 5, HLA-A11 was fully stable in both cell lines when proteasome activity was substantially inhibited. Similar results were

obtained when the cells were treated with z-L3VS and in cells where HLA-A11 was the only HLA-A locus product expressed (not shown). This suggests that the unusual loading of HLA-A11 depends upon the allele itself rather than its specific cellular environment. FIGURE 6. Maturation of HLA-A11 in z-L3VS-treated AF cells. AF cells were treated in the presence or the absence of 10 ␮M z-L VS for 2 h, The maturation of HLA-A11 is not diminished by proteasome 3 metabolically labeled, and chased for 0, 20, 60, or 120 min in the contin- inhibition uous presence of proteasome inhibitor. MHC class I molecules were im- Having noted that the stability of HLA-A11 was independent of munoprecipitated with W6/32 and analyzed by 1D-IEF. The positions of ␤ proteasome activity, we next analyzed whether the subsequent HLA-A1, HLA-B35, HLA-B51, HLA-A11, and 2m are shown on the left. maturation of HLA-A11 and its egress from the ER remained nor- To simplify the comparison of maturation rates, the immature forms of mal. Maturation of individual class I alleles can easily be moni- HLA-A1 and -A11 are indicated by a 0. The 1 and 2 sialylated forms of HLA-A1 and -A11 are indicated by 1 and 2. The identification of the tored by 1D-IEF, since these polypeptides acquire sialic acid mod- mature forms of these alleles is based on numerous previous analyses and ifications as they pass through the trans-Golgi, causing them to the disappearance of mature bands upon sialidase digestion (38) (data not migrate at progressively more acidic (lower) positions in the gel. shown). The maturation of HLA-A1 is retarded by z-L VS, as evidenced ␮ 3 Thus, AF cells were treated with either DMSO or 10 M z-L3VS by the accumulation of the immature form 0. The transport of HLA-B35 and metabolically labeled for 20 min. The cells were chased for 0, and HLA-A11 remains unaffected by proteasome inhibitors. HLA-B51 is

20, 60, or 120 min, and equal numbers of cells were removed at poorly transported in both untreated and z-L3VS-treated cells. 88 CLASS I ALLELES AND PROTEASOME ACTIVITY peptides and exits the ER when the assembly and maturation of differences between family members. HLA-A3 and -A11 both HLA-A1 are severely compromised by the inhibition of protea- strongly prefer a Lys at peptide position 9, unlike the preference some activity. for Arg of HLA-A31 and -A33 (48). HLA-A3 and -A11 also share It should also be noted that the disappearance of immature hypervariable Q62, R114, D116, K144, and R145 amino acids HLA-B35 was not greatly altered by proteasome inhibition (Fig. within the peptide binding groove, a combination of residues that 6), and this was also seen with other cell lines, including OSH and is not shared with HLA-A68, -A31, or -A33 (49). HLA-A3 and HRC-5 (not shown). Along with the observation that the stability HLA-A11 are also located separately from HLA-A31, -A33, and of HLA-B35 did not closely correlate with proteasome activity -A68 on a relatively recent branch of the MHC class I phylogenetic (see Fig. 2B), this suggests that HLA-B35 is an intermediate allele tree (50). Although it remains speculative, these differences could that is only partially refractory to proteasome inhibitors. reflect a more recently evolved capacity to bind peptides engen- dered by a putative protease or peptidase that generates a class I binding product with a C-terminal lysine. Discussion The participation of an as yet unidentified protease or pathway It is widely believed that the proteasome is responsible for gener- in the loading of some MHC class I molecules has been postulated ating antigenic peptides for the MHC class I molecule. In this (26, 51, 52). Such an enzyme could either provide peptides in report we have conducted an allele-specific analysis that chal- parallel to the proteasome or could take over its role in the absence lenges the simple view of peptide supply held to date. Our previous of proteasome function. If such a molecule were to be a trypsin- observations implied that a close correlation exists between pro- like protease that generated products terminating in lysine resi- Downloaded from teasome activity and the peptide loading of MHC molecules that dues, then the loading of HLA-A3 and HLA-A11 could be favored bind peptides harboring a hydrophobic residue at position 9 of the above that of the hydrophobic P9 alleles. Consistent with the idea peptide, especially after stimulation with IFN-␥ (32). These alleles of another protease, we often see a small cohort of lactacystin- or all posses a deep, uncharged F pocket to accommodate bulky z-L3VS-resistant MHC class I molecules (e.g., HLA-A68; Fig. 2) amino acids. This relationship also holds, albeit less well, for at high inhibitor concentrations, when the relationship between HLA-A2, which is partly TAP independent in that it can bind proteasome activity and MHC class I loading breaks down. How- peptides generated in the ER by signal peptidase (42–45). When ever, attempts to block the loading of HLA-A11 and HLA-B35 in http://www.jimmunol.org/ we extended our investigations to encompass two MHC class I AF cells with a variety of other protease inhibitors have proved molecules that bind peptides with basic residues at position 9, and unsuccessful to date (data not shown). thus have an acidic F pocket, no relationship between proteasome Alternatively, a small amount of proteasome function, particu- activity and peptide loading was found. The phylogenetically re- larly of the trypsin-like activity, may be sufficient to provide lated MHC class I molecules, HLA-A3 and HLA-A11, remain enough peptides to fully load HLA-A3 and HLA-A11 and to par- stable in the presence of different proteasome inhibitors, even at tially load HLA-B35. In our experiments, we can never totally very high drug concentrations (Figs. 1, 2, and 4). These conditions inhibit the trypsin-like activity of the proteasome, and it may be

inhibit overall proteasome activity by over 70 to 80%, as judged by that 20% or less of this activity is sufﬁcient to load some MHC by guest on September 24, 2021 assays using ﬂuorogenic peptides. HLA-B35, although not entirely class I molecules that require a basic residue at the C-terminal resistant to proteasome inhibitors, also exhibits a poor relationship position. Residual trypsin-like activity would then not be enough between peptide loading and proteasome activity. All other MHC to load the majority of alleles that require a hydrophobic C termi- class I alleles, even when expressed by the same HLA-A3- or nal residue. It is possible that peptides generated by the trypsin-like HLA-A11-positive cell lines, have a class I loading pattern that activity of the proteasome predominantly bind to HLA-A3 family closely correlates with proteasome activity, as we have previously alleles and that peptides generated by the chymotrypsin-like ac- observed (32). tivity bind to the other alleles. The N-termini of these peptides are

The resistance of peptide loading to proteasome inhibitors is presumably created by a lactacystin/z-L3VS independent and IFN-␥ independent and therefore does not rely upon the level of acidic enzymatic component of the proteasome (11). Specific in- MHC class I expression, alterations in proteasome subunit com- hibitors of either the chymotrypsin-like or trypsin-like activity position, or changes in the peptide profile generated by “immuno- would be required to test this hypothesis directly. proteasomes.” The loading behavior that we observed also cannot In this light, it is intriguing to note that MHC class I molecules be explained by differential affinities for the TAP transporter be- appear to have evolved to fit the products of proteasomal digestion, tween either 1) the C-terminus of the peptide substrate (6) or 2) the with no known MHC class I molecule able to present peptides with MHC class I molecules themselves. Although HLA-A3 and HLA- an acidic residue in peptide position 9 (53). Thus, the relatively A11 interact strongly with TAP, the proteasome-dependent alleles high abundance of alleles requiring a hydrophobic P9 vs a basic P9 HLA-A2, -B51, and -B8 (among others) do so as well (46). could be an evolutionary consequence of the relative abundance of We anticipated that the behavior of HLA-A3 and HLA-A11 these proteasomal products in vivo. might be a common feature of the HLA-A3 superfamily, compris- The finding that proteasome inhibition results in a differential ing HLA-A0301, -A1101, -A3101, -A3301, and -A6801. How- effect on MHC class I loading is novel and intriguing. Inhibiting ever, analysis of cell lines expressing HLA-A68 (e.g., HeLa cells), proteasome function by 70 to 80% has little effect on HLA-A3, -A31, and -A33 alleles has not shown them to withstand protea- -A11, and -B35, while within the same cells, the loading and mat- some inhibition (Fig. 1 and data not shown). HLA-A3 and HLA- uration of all other alleles are severely compromised. These ob- A11 share overlapping peptide specificities and are capable of servations highlight another level of microdiversity in the MHC binding the same peptides with high affinity (47). As well as the class I Ag-processing pathway. characteristic positively charged amino acid at position 9 of the antigenic peptide, A3 superfamily members also prefer a hydroxyl- Acknowledgments containing or hydrophobic residue at position 2. It is unlikely that We thank Dr. N. Lardy (Central Laboratory of the Blood Transfusion Ser- a specific A3 superfamily motif or mutation segregates with pro- vice (CLB), Amsterdam, The Netherlands) for HLA typing of our HeLa teasome unrestricted loading, since only HLA-A11 and HLA-A3 cells, Dr. C. Vos for critical reading of the manuscript, and P. Spee for appear to behave anomalously. However, there are some subtle assistance with the figures. The Journal of Immunology 89

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