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Characterizing the Specificity and Cooperation of Aminopeptidases in the Cytosol and Endoplasmic Reticulum during MHC Class I Antigen Presentation This information is current as of September 29, 2021. Arron Hearn, Ian A. York, Courtney Bishop and Kenneth L. Rock J Immunol 2010; 184:4725-4732; Prepublished online 29 March 2010; doi: 10.4049/jimmunol.0903125 Downloaded from http://www.jimmunol.org/content/184/9/4725

Supplementary http://www.jimmunol.org/content/suppl/2010/03/26/jimmunol.090312 http://www.jimmunol.org/ Material 5.DC1 References This article cites 47 articles, 24 of which you can access for free at: http://www.jimmunol.org/content/184/9/4725.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 © 2010 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Characterizing the Speciﬁcity and Cooperation of Aminopeptidases in the Cytosol and Endoplasmic Reticulum during MHC Class I Antigen Presentation

Arron Hearn,* Ian A. York,† Courtney Bishop,*,1 and Kenneth L. Rock*

Many MHC class I-binding peptides are generated as N-extended precursors during protein degradation by the proteasome. These peptides can subsequently be trimmed by aminopeptidases in the cytosol and/or the endoplasmic reticulum (ER) to produce mature epitope.However,thecontributionandspecificityofeachofthesesubcellularcompartmentsinremovingN-terminalaminoacidsforAg presentation is not well defined. In this study, we investigated this issue for antigenic precursors that are expressed in the cytosol. By systematically varying the N-terminal flanking sequences of peptides, we show that the amino acids upstream of an epitope precursor are a major determinant of the amount of Ag presentation. In many cases, MHC class I-binding peptides are produced through se- Downloaded from quential trimming in the cytosol and ER. Trimming of flanking residues in the cytosol contributes most to sequences that are poorly trimmed intheER.Because N-terminaltrimminghas differentspecificityinthecytosolandER,thecleavageofpeptides inbothofthese compartments serves to broaden the repertoire of sequences that are presented. The Journal of Immunology, 2010, 184: 4725–4732.

ajor histocompatibility complex class I molecules that can then be reused in the synthesis of new proteins (18).

present a large variety of peptides generated by in- However, some escape destruction and are shuttled by the trans- http://www.jimmunol.org/ M tracellular protein degradation to the immune system. porter associated with Ag presentation (TAP) into the endoplasmic This allows the identification and elimination of cells synthesizing reticulum (ER). In addition, some MHC class I-presented peptides abnormal or “foreign” proteins, which may be a result of mutation may be generated through the trimming of long antigenic precursors or infection by virus (1–5). in the cytosol (19). TAP translocates peptides longer than 16 resi- Peptides that can stably bind MHC I molecules are generally 8–10 dues inefficiently in vitro (20); therefore, trimming of long pre- residues in length and are generated by a complex pathway in- cursors by peptidases within the cytosol may also be critical for the volving many components in several cellular compartments. The generation of peptides suitable for TAP binding (21). first step in this pathway is degradation of proteins by the protea- A substantial amount of the trimming of N-extended precursors some in the nucleus or cytosol. This multisubunit protease is capable is performed in the ER by the IFN-g–inducible metallopeptidase by guest on September 29, 2021 of generating mature epitopes (∼ ,5% of product) (6). However, the ER aminopeptidase 1 (ERAP1) (ER aminopeptidase associated majority of epitope-containing peptides (15–25% of proteasome with Ag processing) (19, 22, 23). ERAP1 prefers substrates 9–16 product) (6) are extended on the N terminus and are incapable of residues long (19, 24), which corresponds to the length of peptides MHC binding unless processed further (7). A large number of efficiently transported by TAP (20). ERAP1 seems to be particu- aminopeptidases and endopeptidases within the cytosol have the larly adapted to generate peptides for MHC class I presentation, potential to create or destroy MHC-binding peptides from the pre- because it rapidly trims precursors down to eight or nine residues cursors generated by the proteasome (8–17). Within seconds, the before cleavage slows or stops completely. Thus, unlike other majority of peptides in the cytosol are hydrolyzed into amino acids aminopeptidases that continually cleave peptides until only one free amino acid remains, ERAP1 acts with a molecular ruler to generate peptides of the optimal length for MHC class I binding *Department of Pathology, University of Massachusetts Medical School, Worcester, (24). It was also suggested that ERAP1 cooperates with MHC MA 01655; and †Department of Microbiology and Molecular Genetics, Michigan class I molecules to generate mature epitopes, although the State University, East Lansing, MI 48824 available data do not distinguish whether MHC class I molecules 1 Current address: David H. Smith Center for Vaccine Biology and Immunology, are actually involved in the trimming process or only bind and University of Rochester, Rochester, NY. protect mature epitopes from destruction (25). In cells lacking Received for publication September 25, 2009. Accepted for publication February 28, 2010. ERAP1, many antigenic precursors are trimmed poorly, if at all, in the ER (19, 22, 26–28); mice lacking ERAP1 had markedly dif- This work was supported by Grant R01 AI20248-27 from the National Institutes of Health (to K.L.R.) and core resources were supported by the Diabetes Endocrinology ferent presentation of a variety of epitopes (26–29), leading to Research Center Grant 5 P30 DK32520. differences in immunodominance hierarchies (26, 29, 30). How- Address correspondence and reprint requests to Dr. Kenneth L. Rock, Department of ever, precursor peptides generated in the cytosol are still presented Pathology, University of Massachusetts Medical School, Worcester, MA 01655. to various extents in ERAP1-deficient cells, indicating that some E-mail address: [email protected] trimming of presented peptides occurs in the cytosol (19). The online version of this article contains supplemental material. Schatz et al. (31) recently investigated the role that the ER and Abbreviations used in this paper: ER, endoplasmic reticulum; ERAP1, endoplasmic reticulum aminopeptidase 1; MEF, mouse embryonic fibroblast; MFI, mean fluores- cytosol may play in epitope generation in cell extracts, and we cence intensity; S-L, chicken ovalbumin peptide SIINFEKL; siRNA, small interfer- examined the role of ER trimming in intact cells (32). These ing RNA; ss, signal sequence; TAP, transporter associated with Ag presentation; TOP, studies suggest that trimming in the ER plays an important part in thimet oligopeptidase; Ub, ubiquitin. generating antigenic peptides, but it does not account for all Copyright Ó 2010 by The American Association of Immunologists, Inc. 0022-1767/10/$16.00 peptide processing. Understanding the contribution and specificity www.jimmunol.org/cgi/doi/10.4049/jimmunol.0903125 4726 SPECIFICITY OF CYTOSOLIC AND ER AMINOPEPTIDASES of nonproteasomal proteases in vivo in the generation of CTL epitope, generating XXS-L, where X represents any amino acid. epitopes should enhance our understanding of epitope generation These sequences were ligated in frame to the 39 region of ubiquitin and modeling of this process. This led us to initiate the current (Ub) so that they would be expressed as fusion proteins (Ub XXS-L) study to systematically examine the specificity of trimming N- in the cytosol. Ub C-terminal hydrolases in cells efficiently and pre- terminal sequences from antigenic precursors in vivo. cisely cleave the peptide bond between the Ub moiety and any fused sequence [unless the residue flanking the scissile bond is a proline Materials and Methods (35), which is why proline was omitted from our series]; thus, when expressed in cells, these constructs efficiently generate XXS-L in the Plasmids cytoplasm and avoid the need for an initiating methionine on the N To express N-terminal SIINFEKL (OVA254–264) (S-L) precursors in the terminus of the antigenic peptide. The Ub XXS-L sequences were cytoplasm, synthetic complementary oligonucleotide primers (Integrated cloned into a plasmid that bicistronically expressed GFP,allowing the DNA Technologies, Coralville, IA) were annealed and inserted into the direct correlation of GFP and Ub XXS-L expression. plasmid pUG-1 (11). This generates a plasmid consisting of Ub with (X)n–S- L (where n is a variable number of residues) fused to the C terminus. The The Ub XXS-L constructs were transiently transfected into HeLa- oligonucleotides for cytoplasmic expression were, for Ub-XXS-L, 59-XXX Kb cells, and the amount of S-L-Kb complexes on the cell surface of XXX TCC ATC ATC AAC TTC GAG AAG CTG TAA TAGCTGCAG- cells expressing similar levels of GFP was quantified by staining GATCC-39 and for Ub-4N-X-S-L (Ub-LEQLXS-L), 59-CTG GAG CAG with the mAb 25.D1.16, which recognizes the combination of H- CTG XXX TCC ATC ATC AAC TTC GAG AAG CTG TAA TAGCTG- b CAGGATCC-39. A PstI site (CTGCAG) was used for selection. XXX is 2K and S-L (34). When N-extended precursor peptides are ex- b a codon that encodes for an amino acid. The most common codons for each pressed in cells, only the mature epitopes are presented on H-2K amino acid, based on the frequency of usage of each codon (per thousand) in and detected by 25.D1.16 (19, 21). This assay gave highly re- Downloaded from human coding regions, were used: ala (A), GCT; arg (R), CGC; asn (N), producible results between replicate groups and independent ex- AAC; asp (D), GAC; cys (C), TGT; gln (Q), CAG; glu (E), GAG; gly (G), GGC; his (H), CAC; ile (I), ATC;leu (L), CTG; lys (K), AAG; met (M), ATG; periments. This analysis revealed that N-terminal flanking amino phe (F), TTC; ser (S), TCC; and thr (T), ACC. All plasmids were sequenced acids had a marked influence on the levels of presented peptide to confirm correct sequences and reading frames. Generation of signal se- detected on the cell surface (Fig. 1A). S-L from all constructs was quence (ss) XXS-L plasmids were described previously (32). presented, but at different levels, such that there was a highly re-

Cells producible hierarchy of presentation that depended on the specific http://www.jimmunol.org/ N-terminal residues. Amino acids, such as alanine, cysteine, tyro- HeLa-Kb and HeLa-Kb-ICP47 (HeLa-Kb-A3-47 cells [HeLa cells stably sine, and methionine, seem to be removed efficiently from pre- transfected with H-2Kb, HLA-A3 and ICP47]) cells were described previously (19, 33). Mouse embryonic fibroblasts (MEFs) were prepared from cursors to generate the mature S-L epitope. In fact, 16 of the 19 aa wild-type C57BL/6 (two independent lines) and ERAP1 knockout mice (including alanine) were associated with presentation that was (two independent lines), as previously described (26). $40% of that observed with Ub AAS-L (the construct leading to the greatest presentation), demonstrating that many amino acids are Small interfering RNA removed rather efficiently. Only glutamate, aspartate, and glycine Cells were transfected with small interfering RNA (siRNA) for ERAP1 or were associated with lower presentation: ,40% of that observed with the control siRNA murine thimet oligopeptidase (TOP) (directed with Ub AAS-L. However, although the acidic amino acids gluta- by guest on September 29, 2021 against murine TOP in a region that differs from human TOP) using oli- gofectamine (Invitrogen, Carlsbad, CA) as previously described (19). mate and aspartate seem to be associated with low presentation, the hierarchy of presentation did not otherwise correlate with the Abs and flow cytometry chemical nature of the side chain. Thus, among the various classes of HeLa cell lines were transiently transfected with plasmid (0.25–1 mg) using amino acids (e.g., basic, hydrophobic, etc.), there was a broad range TransIT HeLa Monster (Mirus, Madison, WI), according to the manu- of efficiencies of presentation within each group (Fig. 1B). facturer’s protocol. MEFs were transfected with FuGene6 (Roche, In- If the Ub C-terminal hydrolases were to cleave the various Ub dianapolis, IN), according to the manufacturer’s protocols. After XXS-L constructs imprecisely or at different rates, this could con- transfection, cells were incubated for 24–48 h, and H-2Kb-S-L complexes on the cell surface were detected using mAb 25.D1.16 (34). The cells were tribute to the differential presentation of the various Ub XXS-L Ags. analyzed by flow cytometry on a FACSCalibur apparatus (BD Biosciences, Although these proteases are thought to cleave precisely and in- San Jose, CA) with CellQuest (BD Biosciences) or FlowJo (Tree Star, dependently of the P9 residue (35),we soughtto evaluate this issue. In Ashland, OR) software, gating on GFP-expressing cells to limit the anal- an attempt to exclude the possibility that differences in presentation ysis to transfected cells (Supplemental Fig. 1). Unless otherwise stated, data are representative of three independent experiments. between the various constructs was due to the specificity of the Ub hydrolases, we sought to generate XXS-L in another way. For these experiments, we generated a subset of minigenes that consisted of Results a Kozac consensus sequence followed by an initiating methionine, The effect of N-terminal flanking residues on the presentation and the sequence AXXS-L (we excluded X = M from this analysis of cytosolic precursors because of the potential for multiple translation initiation sites) were Because antigenic epitope precursors are first generated in the X-encoded amino acids that were processed efficiently (L and N) cytosol, which contains resident aminopeptidases that can trim and inefficiently in the cytoplasm (E, G, and I), as well as amino peptides, we sought to analyze the specificity of trimming of N- acids that lie between these two extremes (F, K, Q, and V). When extended precursors in this compartment. these constructs were transfected into HeLa-Kb cells, they were To systematically examine the specificity of trimming of N-ex- trimmed and presented on H-2Kb (Fig. 1C). Again, there was a hi- tended precursors of MHC class I-presented peptides, we generated erarchy of presentation of the various constructs; importantly, this a series of minigene constructs containing the model epitope SIIN- hierarchy was essentially the same as that observed with Ub XXS-L FEKL (S-L) (the immunodominant H-2Kb–restricted epitope from (Fig. 1C). The concordance of the data from the Ub XXS-L and chicken OVA). Minigenes were constructed to encode 19 different MAXXS-L constructs argues strongly that the hierarchy in pre- amino acids (proline was excluded for reasons described below) on sentation is not due to differences in the generation of precursors but the N terminus of the mature epitope. To amplify any differences rather is due to processing of the precursors to the mature peptide. between constructs in the rate of removal of their flanking residues, Given that the identity of the amino acid repeated twice N- they were expressed with two identical amino acids upstream of the terminally of an epitope markedly influences its presentation when The Journal of Immunology 4727

FIGURE 1. Ag presentation in HeLa-Kb cells transfected with cytoplasmic targeted N-terminal S-L Downloaded from precursors. Cells were transfected with Ub XS-L, Ub XXS-L, or MAXXS-L (A, inset), where X represents one of the 20 aa. H-2Kb-S-L presentation was determined by staining cells with 25.D1.16. Data are arranged in order of efﬁciency of presentation, from highest to lowest, left to right (A) or are grouped ac- http://www.jimmunol.org/ cording to the chemical nature of the amino acid side chain (B). Bar graphs represent the average mean ﬂuorescence intensity (MFI) of two independent transfections. Error bars represent the difference within each group. Correlation of S-L presentation from Ub XXS-L and MAXXS-L (C) or Ub XS-L (D). by guest on September 29, 2021

expressed in the cytosol, we next examined whether a single amino aminopeptidase ERAP1 seems to be the only important peptide- acid showed the same effect as the corresponding doublet. To this trimmingenzymaticactivityintheERofHeLacells,asdemonstrated end, we generated a subset of Ub XS-L constructs that encoded using S-L precursors with the natural flanking sequence (LEQLE) amino acids that were processed efficiently (L, M, and Y) and (19, 22). Therefore, we sought to determine how much ERAP1 inefficiently in the cytosol (D, E, and G), as well as amino acids that contributes to the presentation of the various Ub XXS-L constructs. lie in between these two extremes (K and R). The data obtained These precursors were transfected into HeLa-Kb cells treated with with these constructs (Fig. 1D) are largely consistent with those ERAP1-specific siRNA under conditions in which ERAP1 protein obtained with the corresponding doublet, although the absolute expression is reduced by $90% (19), and presentation was com- differences in the level of presentation were smaller, presumably pared with cells treated with a control irrelevant siRNA. due to the reduction in the number of residues that needed to be Ub C-terminal hydrolase cleavage of the control Ub S-L in removed to generate mature epitope (Fig. 1A). the cytosol generates mature S-L directly. Because this mature epitope requires no ER trimming for presentation, its presentation Ub XXS-L trimming by ERAP1 should not be dependent on ERAP1. Treatment with ERAP1 siRNA Antigenic precursors produced in the cytosol could potentially be did not affect presentation from this precursor (Fig. 2A), demon- trimmed in the cytosol and/or in the ER after transport by TAP. The strating that the siRNA does not cause off-target effects on Ag 4728 SPECIFICITY OF CYTOSOLIC AND ER AMINOPEPTIDASES

FIGURE 2. Effect of ERAP1 knockdown on S-L presentation from cytosol- targeted XXS-L. HeLa-Kb cells were treated with siRNA targeting ERAP1 or control siRNA and then transfected with Ub XXS-L. S-L presentation was determined on day 3. A, FACS showing cells stained with 25.D1.16 following transfection with Ub S-L, Ub GGS-L, and Ub WWS-L; cells treated with control siRNA (solid line) or ERAP1 siRNA (dashed line). Shaded curve represents back- ground. B, Percentage of S-L presentation that is ERAP1 dependent, following HeLa cells transfected with Ub XXS-L. Ar- Downloaded from ranged from highest to lowest, left to right. Calculated from the MFI of cells treated with control (100%) or ERAP1 siRNA. Bar graphs represent the average of two independent experiments. C, ERAP1 dependence of Ub XXS-L constructs transfected into MEFs (ERAP12/2 http://www.jimmunol.org/ MEFs compared with ERAP+/+ MEFs) arranged from highest to lowest (graphs represent the average percentage of three independent experiments). D, Scatter graph showing the correlation of ERAP1 dependence between MEFs and HeLa cells. by guest on September 29, 2021

presentation. In contrast, the presentation of S-L from most of the 2C), and the pattern of dependence on ERAP1 correlated strongly Ub XXS-L constructs was reduced in ERAP1-silenced cells, between MEFs and HeLa cells (R2 = 0.82) (Fig. 2D). The mag- suggesting a role for this enzyme in epitope generation from cy- nitude of ERAP1 dependence in MEFs was greater than in HeLa tosolic precursors. Interestingly, there was a range of ERAP1- cells, ranging from ∼30% to nearly 100%, suggesting that the dependent presentation from these various N-terminally extended small amounts of ERAP1 in siRNA-treated HeLa cells may con- precursors. For example, presentation from Ub GGS-L was se- tribute slightly to peptide processing. Alternatively, MEFs may verely reduced in ERAP1-silenced cells, whereas presentation have a slightly different spectrum of peptidases, such that ERAP1 from Ub WWS-L was unaffected (again, a finding suggesting no is more important than in HeLa cells. In any case, the strong off-target effects of the siRNA-treatment) (Fig. 2A). Dependence correlation between HeLa cells and MEFs suggests that peptide on ERAP1 was approximately similar for the other 17 constructs processing is remarkably similar between different cell types, as and fell between these two extremes (Fig. 2B). Overall, the well as between different species. magnitude of the ERAP1 dependence ranged from 0–80%. Interestingly, even in MEFs, the absence of ERAP1 only The presentation that occurs in ERAP1-silenced cells pre- modestly reduced the presentation of the Ub WWS-L construct. sumably reflects trimming occurring in the cytosol and possibly Therefore, a cytosolic peptidase can play a dominant role in the some contribution from residual ERAP1. The large difference in removal of tryptophan residues upstream of S-L prior to transport ERAP1 dependence of the various XXS-L constructs suggests that into the ER. there is sequence specificity for trimming by other peptidases responsible for the generation of S-L. Although ERAP1 siRNA Presentation and ERAP1-dependence of precursors expressed reduces ERAP1 expression by $90% (19), a small amount of in the cytosol versus the ER residual ERAP1 could still process precursors under these con- It was of interest to compare the presentation of XXS-L constructs ditions. To test the potential contribution of residual ERAP1, we in the cytosol versus ER. To do this, we compared the presentation measured the presentation of a subset of peptides in MEFs pre- of the Ub XXS-L peptides with ER-targeted peptides that we had pared from wild-type (ERAP1+/+) or ERAP1 knockout mice, in previously generated and characterized in HeLa-Kb cells stably which ERAP1 is completely absent. Again, the dependence on transfected with ICP47 (32). ICP47 blocks the TAP transporter and ERAP1 for presentation was different for different constructs (Fig. prevents cytosolic peptides from gaining access into the ER (36), The Journal of Immunology 4729 allowing specific analysis of ER-specific trimming. These ER- specificities of the aminopeptidase(s) in this compartment (32). targeted constructs consisted of the XXS-L sequences that were ERAP1 seems to play a major role in ER precursor editing in HeLa fused to an N-terminal signal sequence (ss XXS-L). Upon syn- cells stably transfected with ICP47 (32), as well as in many cases thesis, these constructs are transported via the sec61 translocon of cytosolic precursors in HeLa cells with an active TAP transporter into the ER, where the leader peptide is removed by the signal (Fig. 2). We next compared the role of ERAP1 in the processing of sequence peptidase; our previous studies found that differences in cytosolic precursors (Fig. 2) with that of ER precursors in HeLa the presentation of these constructs were not due to differences in cells with an active TAP transporter. Fig. 3C shows that all of the efficiency of cleavage of the various constructs by the signal the ER-targeted precursors (including WWS-L) rely much more sequence peptidase (32). heavily on the activity of ERAP1 than do the majority of the cy- We previously examined the presentation of the ER-targeted tosolic precursors. Thus, a large percentage of presentation from constructs in HeLa-Kb cells in which TAP was inhibited (32). In cytosolic precursors is due to an ERAP1-independent component, HeLa-Kb cells with a functional TAP transporter, the difference and the differences in presentation efficiency must reflect trimming between highest and lowest presentation of Ub XXS8L and ss within the cytosol. XXS8L constructs was much greater for the ER-targeted pre- Presentation from precursors with and without TAP activity cursors than for the cytosolic precursors, and presentation from many of the epitope precursors was greater in relative terms if To further assess the compartment in which the various cytosolic or expressed in the cytosol than if targeted to the ER (Fig. 3A). As ER precursors are trimmed, we compared the presentation of S-L in a result, when the averages of the results obtained in Fig. 3A were HeLa-Kb or HeLa-Kb-ICP47 (HeLa cells stably transfected with plotted, there was a poor correlation between presentation of the ICP47) cells (33). No presentation was observed when SIINFEKL, Downloaded from same constructs targeted into ER compared with the cytosol (Fig. the mature epitope, was expressed within the cytoplasm (Ub S-L), 3B). Presentation from cytosol precursors is probably the result of indicating efficient TAP inhibition, whereas ER-targeted S-L (ss a combination of editing in the cytosol and the ER (Fig. 2), S-L), which bypasses the need for TAP transport, was presented whereas processing of ER precursors is only dependent on the efficiently (Fig. 4A). As expected, the presentation of all of the cytosolic precursors (Ub XXS-L constructs) was essentially http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 3. Comparison of precursors expressed in the cytosol and the ER. A, HeLa-Kb cells were transfected with ER- (ss; black bars) or cytosol- FIGURE 4. Effect of TAP inhibition on S-L presentation from cytosol (Ub; white bars) targeted XXS-L and stained with 25.D1.16. Data are ar- and ER precursors. A, HeLa-Kb-ICP47 cells were transfected with Ub ranged in order of efﬁciency of presentation from ER- targeted precursors XXS-L for 24 h and stained for surface H-2Kb-S-L. ER-targeted S-L (ss S- from highest to lowest, left to right. B, Correlation of S-L presentation from L) and cytoplasmic S-L (Ub S-L) were used as a positive control and Ub XXS-L and ss XXS-L (from the mean data in A). Following treatment negative control, respectively. B, Comparison of S-L presentation from with control or ERAP1 siRNA, HeLa-Kb cells were transfected with ss ER-targeted precursors following transfection of HeLa-Kb cells (black XXS-L or Ub XXS-L. C, The percentage of S-L presentation that is ERAP1 bars) and HeLa-Kb-ICP47 cells (white bars). Bar graphs represent the dependent was calculated [100 2 (MFI with ERAP1 siRNA/MFI with average percentage presentation of two independent wells using the MFI of control siRNA) 3 100]. The Ub XXS8L data in C are from Fig. 2B and are cells transfected with ss MMS-L as 100% presentation. Error bars repre- regraphed for comparison with the new ss XXS8L data. Data represent the sent the difference within each group. C, Using the data in B, the per- average percentage of two independent experiments centage TAP dependence of each ss XXS-L construct was calculated. 4730 SPECIFICITY OF CYTOSOLIC AND ER AMINOPEPTIDASES completely inhibited in HeLa-Kb-ICP47 cells, because these cytosolic peptides require TAP for transport into the ER. In contrast, the presentation of S-L from most ER-targeted precursors was efﬁcient (Fig. 4B). Unexpectedly, however, the presentation of S-L from many ER-targeted constructs (e.g., those with D, W, and E) was partially to almost completely inhibited by ICP47 (Fig. 4B, 4C), implying that the presentation of these ER-targeted precursors remained at least partially dependent on TAP.

X/XXS-L cytosol dependence Several studies have raised the possibility of retrotranslocation of epitope precursors from the ER into the cytoplasm for trimming, followed by reimport of peptides into the ER for presentation (37– 39). In this study, some of the ER-targeted precursors (ss XXS-L) that show a strong reliance on the cytosol for presentation (i.e., are inhibited by ICP47; Fig. 4B), also require ERAP1 for processing (Fig. 3C). This suggests that epitope production may be dependent on trimming in the cytosol and ER. Assuming that the ER-targeted construct is not mistargeted into the cytosol, the epitope precursor Downloaded from must be retrotranslocated into the cytoplasm for processing. Thus far, our findings suggest that for ER-targeted precursors that require ERAP1 and TAP, these XXS-Ls are poor substrates for ERAP1 and must be converted into XS-L in the cytosol (XXS-L → XS-L) for ERAP1-dependent removal of X (XS-L→S-L) or that following the ERAP1-dependent generation of XS-L in the FIGURE 5. ER-targeted precursors require cytosol trimming. A, HeLa- http://www.jimmunol.org/ ER (XXS-L → XS-L), S-L must be generated in the cytosol (XS- Kb cells (black bars) and HeLa-Kb-ICP47 cells (white bars) were trans- L→S-L), because these XS-Ls are poor substrates for ERAP1. To fected with ss XS-L or ss XXS-L. Bar graphs represent the average per- distinguish between these possibilities, we compared the pre- centage presentation of two independent wells using the MFI of cells sentation from ss D/W/ES-L and ss DD/WW/EES-L, the latter of transfected with ss MMS-L as 100% presentation. Error bars represent the which is dependent on ERAP1 and the cytosol for presentation difference within each group. B, HeLa-Kb cells were treated with siRNA targeting ERAP1 (white bars) or ctrl siRNA (black bars) and transfected (Figs. 3C,4B,4C) in HeLa-Kb cells with and without ICP47. ss with ss XS-L or ss XXS-L. Bar graphs represent the average percentage MS-L and ss MMS-L, the latter of which is ERAP1 dependent and presentation of two independent experiments using the MFI of cells treated cytosol independent (Figs. 3C,4B,4C), were used as controls. with control siRNA and transfected with Ub MMS-L as 100% pre- by guest on September 29, 2021 Presentation from ss MS-L and ss MMS-L was not TAP de- sentation. Error bars represent the difference within each group. pendent, implying that M and MM can be removed efficiently in the ER for S-L production (Fig. 5A). Interestingly, although presentation from ss DD/WW/EES-L was inhibited when TAP was inactivated, as shown previously, the presentation from ss W/ES-L The specificity of trimming in these compartments in vitro has and, to a lesser extent, ss D S-L was not affected (Fig. 5A). This been studied using fluorescent substrates and purified cytosol or ER suggests that the W/E residue can be efficiently removed from W/E (31). However, in vivo, the effect that sequences N-terminal to an S-L in the ER, without the need for any other component. To test epitope have on processing and presentation on MHC class I this further, precursors were transfected into HeLa-Kb cells that molecules has only been systematically studied in the ER (32). were treated with control or ERAP1-specific siRNA (Fig. 5B). S-L This is important to define for cytosolic trimming, because ex- presentation was reduced in ERAP1 knockdown cells transfected tracts may not faithfully reproduce the conditions in living cells with ss D/W/E/M S-L as well as ss DD/WW/EE/MMS-L, dem- (e.g., concentrations and ionic conditions are changed, enzymes onstrating that ERAP1 was required for removal of the N-terminal may be activated or inactivated, and metabolic pathways are in- amino acids in all of these precursors. Taken together, this is sug- hibited); presumably, because of this, the specificity of trimming gestive of a mechanism that involves XXS-L retrotranslocation into that we and Reits et al. (9) observed in vivo is not identical to that the cytoplasm for XS-L generation, followed by transport back into reported by Schatz et al. (31). Defining what is occurring in vivo is the ER for ERAP1-specific generation of S-L. important biologically, because the specificity of trimming can clearly influence the magnitude of responses and overall immunodominance hierarchies. In this study, we analyzed the trim- Discussion ming of precursors in the cytosol of living cells and compared it Proteasomes generate many N-terminally extended precursors. For with the trimming of the same precursors in the ER. these precursors to be presented on an MHC class I molecule, they Our experimental approach was to express, in living cells, N- must be trimmed by peptidases to mature epitopes. Previous studies extended precursors in which we systematically varied the amino showed that the removal of amino terminal flanking residues occurs acids at the P2 and/or P1 position N-terminal to the SL8 epitope. Our in living cells. This N-terminal trimming was “downstream” (in- findings clearly demonstrate that trimming of these precursors can dependent) of proteasomes and mediated by aminopeptidases. It occur in the cytosol and in the ER; the efficiency of cytosolic- was clear that this peptide editing could, in principle, occur in the trimming process, like that of ER trimming (32), is affected by the N- cytosol, where peptides were first generated by proteasomes, and/ terminal residues, i.e., it has specificity; the specificity of cytosolic or after transport into the ER by TAP. However, the relative trimming is distinct from that in the ER; recycling of peptides from contribution of these two compartments to trimming and their the ER to the cytosol may occur, potentially allowing sequential specificity had been incompletely understood. trimming of peptides in both compartments in either order; and the The Journal of Immunology 4731 net effect of cytosolic trimming is to broaden the repertoire of presented equally well in control cells and cells in which TAP peptides that can be presented on MHC class I molecules. was inhibited with ICP47, as expected. However, a remarkable Our experimental approach makes certain assumptions that are finding was that the presentation of some ER-targeted precursors worth discussing. We expressed a series of peptides from minigenes was strongly inhibited by ICP47. It is possible that these were that were transfected into APCs. Our interpretation of the results ones that failed to translocate through SEC61. However, this assumes that the transcription, translation, and, for Ub-X con- explanation seems unlikely, because SEC61 can clearly transport structs, the posttranslational Ub cleavage, are similar for all con- the particular residues when they are in other locations (e.g., E in structs. Because the Ub construct bicistronically expresses GFP, we S-L) or even when the residue is present as a singlet (ss XS-L), are able to gate on cells expressing similar levels of GFP that should and SEC61 obviously transports proteins that have all 20 aa. also be expressing similar levels of the Ub fusion proteins. To Another possibility is that the signal peptidase failed to cleave the further test this assumption, we compared presentation from signal peptide from certain precursors, and these had to be minigene constructs that are processed very differently and retrotranslocated to the cytosol for the signal peptide to be re- obtained very similar results using MAXXSL, Ub-XXS-L, and Ub- moved. However, whether cleavage occurs depends mainly on XS-L constructs. This rules out the possibility that differences in features of the signal peptide that remain unchanged in all of the presentation arise from differential Ub -X cleavage and makes it constructs used throughout this study, particularly the amino highly unlikely that one (X) or two (XX) residues placed at dif- acids at positions 23and21 N-terminal of the cleavage site ferent locations (2 or 76 residues) from the translational start site (reviewed in Ref. 46). It seems more plausible that these se- would affect translation (or transcription) and do so in the exact quences need export and trimming in the cytosol because they same way. Therefore, differences in translation, transcription, and areverypoorlytrimmedintheER.Indeed,theresiduesthatare Downloaded from or Ub cleavage are unlikely to account for the differences in ICP47 inhibitable are the ones that are poorly removed by presentation that were seen with different specific sequences. ERAP1 (32) and are particularly poorly removed when they are Although it is possible that these upstream residues may also af- present in tandem. fect TAP transport of cytosolic precursors, the residues that are Several previous studies raised the possibility of export followed consistently associated with high-level presentation of our model by reimport of peptides into the ER for presentation. For example,

epitope are not particularly preferred for TAP translocation (40, in vivo, epitopes are generated in a TAP-dependent mechanism http://www.jimmunol.org/ 41). Moreover, we saw very similar results in mouse and human from the signal sequence of LCMV gp33 protein (37), as are HLA- cells, although mouse and human TAP have somewhat different E–binding epitopes generated from the signal sequences of MHC preferences for translocation (42–45). Therefore, we interpret that class I molecules (38). Such TAP-dependent presentation could be presentation observed with the various epitope precursors reflects due to failed translocation of the signal sequence into the ER or aminopeptidase specificity within the cell. However, it should be retrotranslocation of peptide precursors from the ER into the cy- pointed out that, even if TAP selectivity contributes to some of the tosol. An elegant study by Altrich-VanLith et al. (39) showed this observed differences, our results still define the overall specificity to be the case for an epitope derived from tyrosinase. In this of Ag presentation within the cell (i.e., cytosolic trimming, TAP system, TAP inhibition significantly diminished presentation from transport, and ER trimming). In addition, for the ER-targeted ER-targeted precursors with two-residue extensions containing by guest on September 29, 2021 constructs we assume that XX residues do not influence cleavage histidine. His is one of the amino acids poorly removed in the ER, by the signal peptidase, which liberates the epitope precursor from and it required cytosolic trimming in our experimental system (ss the signal peptide. This is supported by two pieces of data that HHS-L). In addition, earlier studies of isolated microsomes also show that the same results are obtained for sequences that are demonstrated that ER luminal peptides could be retrotranslocated adjacent (ss X-S-L) or six residues away (ss LEQLXS-L) from the out of the ER and recycle back in a TAP-dependent fashion (47). signal sequence cleavage site and that presentation from mini- Thus, there is growing evidence that ER-to-cytosol peptide genes and trimming of the same sequences by purified ERAP1 translocation can occur, although the biological importance of this correlate well with one another (32). phenomenon was unknown. In these earlier studies, it was unclear Our data show that much of the trimming of precursor peptides why retrotranslocation might be important for presentation. Our occurs in the ER, even when the peptides are originally generated in findings suggest that ER-to-cytosol recycling may be important in the cytosol, but the relative importance of ER versus cytosolic situations in which ERAP1 does not trim particular sequences trimming depends on the specific N-terminal residues. In one case, down to mature epitopes. Our data further suggest that recycling extensive trimming of a cytosolic precursor can occur in the cytosol of peptides between the ER and cytosol is likely to be a general (WWS-L). This N-terminal flanking sequence must be removed phenomenon that occurs physiologically and contributes to MHC very efficiently in the cytosol, because when this same construct class I Ag presentation. was targeted into the ER directly, it was efficiently trimmed by In conclusion, our findings reveal that the amount of MHC– ERAP1. In general, cytosolic trimming is more important for those peptide complex presented to the immune system is determined by sequences that are more poorly trimmed in the ER, broadening the the identity of amino acids upstream of the epitope precursor and sequences that can be efficiently processed and presented. As the ability of aminopeptidases to remove them. In many cases, a result, the difference between the best and the worst presented mature epitope generation is a combination of processing that has constructs was smaller when N-extended peptides were expressed occurred in the cytosol and the ER. Taken together, this demon- in the cytosol compared with the ER. Only N-terminal glycine was strates an important role for aminopeptidases in influencing the associated with relatively poor presentation in cytosolic- and ER- specificity of CD8+ T cell responses. targeted constructs. Nevertheless, the trimming that occurs in the cytosol does have specificity. There is a reproducible hierarchy, observed in human and mouse cells, in the trimming and pre- Acknowledgments sentation of the various constructs based on the identity of their N- We thank Sopia Zendzian for technical assistance. terminal residues. Many of the precursors that were targeted by a signal sequence Disclosures through SEC61 into the ER that were efficiently trimmed were The authors have no financial conflicts of interest. 4732 SPECIFICITY OF CYTOSOLIC AND ER AMINOPEPTIDASES

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