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The Journal of Immunology, 2008, 181, 6275 -6282
Copyright © 2008 by The American Association of Immunologists, Inc.
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Endoplasmic Reticulum Aminopeptidase Associated with Antigen Processing Regulates Quality of Processed Peptides Presented by MHC Class I Molecules1

Takayuki Kanaseki and Nilabh Shastri2

Division of Immunology, Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Effective immune surveillance by CD8 T cells depends on the presentation of diverse peptides by MHC class I (pMHC I) molecules on the cell surface. The pMHC I repertoire is shaped in the endoplasmic reticulum (ER) by the ER aminopeptidase associated with Ag processing (ERAAP). The ERAAP activity is required for producing peptides of appropriate length for generating optimal pMHC I. Paradoxically, ERAAP also inhibits generation of certain peptides such as the SVL9 (SSVVGVWYL) peptide encoded by the H13a histocompatibility gene and presented by Db MHC by an unknown mechanism. In this study, we show that the presentation of the SVL9-Db complex is inhibited when other peptides compete for binding Db. Conversely, improving the binding of SVL9 peptide to Db suppresses the inhibition. Interestingly, the inhibitory effect of competitor peptides is observed only when ERAAP is expressed in the same cells. Thus, ERAAP, in concert with MHC I molecules, regulates the quality of processed peptides presented on the cell surface.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The cytolytic CD8 T lymphocytes identify their targets by the presence of novel peptides presented by MHC class I molecules (pMHC I)3 on the cell surface (1, 2). When normal cells are infected with microbes or transformed into tumor cells, peptides from microbial or mutant proteins are also loaded onto MHC I molecules. These new pMHC I serve as potential ligands for eliciting a CD8 T cell response, which can eventually eliminate the abnormal cells. The pMHC I repertoire, generated by the Ag processing pathway, is therefore essential for immune surveillance by the CD8 T cell repertoire.

The Ag processing pathway begins with proteolysis of intracellular proteins in the cytoplasm (3). The proteolytic fragments are transported into the endoplasmic reticulum (ER) by TAP (4). In the ER, an aminopeptidase called ERAAP (ER aminopeptidase associated with Ag processing) trims the N-termini of most peptides that are chaperoned by the MHC I to the surface of the APCs (5, 6). The pMHC I complexes on the cell surface, available for recognition by the CD8 T cells, are therefore a close reflection of the final ER events in the Ag processing pathway (7).

In the ER, the pMHC I repertoire is shaped by ERAAP (5, 6, 8). ERAAP deficiency in mice disrupts the normal pMHC I repertoire significantly as well as CD8 T cell responses (9, 10, 11, 12, 13). Measurement of individual peptides in the pMHC I repertoire of ERAAP-deficient compared with wild-type cells has shown that while some peptides remain unchanged, other peptides such as the WI9 peptide derived from the Y-chromosome encoded Uty gene are no longer detected (10). Because ERAAP, as an aminopeptidase, trims the N-terminal residues flanking the final antigenic peptides in the ER, precursors arriving from the cytoplasm without any extra N-terminal flanking residues would not require ERAAP trimming for presentation as pMHC I (5, 10). In contrast, peptides containing extra N-terminal residues do require ERAAP-trimming, which could explain the absence of these pMHC I in ERAAP-deficient cells. These ERAAP-dependent pMHC I are not available to induce tolerance in the CD8 T cell repertoire of ERAAP-deficient mice. The ERAAP-deficient mice, therefore, respond to the ERAAP-dependent pMHC I when immunized with wild-type cells (9).

Intriguingly, another set of peptides was found to be over-expressed in ERAAP-deficient cells (10, 11). APCs from ERAAP-deficient mice were significantly superior to those from wild-type mice in activating T cells specific for endogenous peptides derived from the H47a or H13a histocompatibility genes (14, 15). Quantitative measurements of the H13a-derived peptide (SSVVGVWYL, SVL9) presented by Db MHC revealed that SVL9 peptide was expressed at least 100-fold higher amount in ERAAP-deficient compared with wild-type cells (10). The presence of ERAAP was, therefore, clearly deleterious to presentation of the SVL9 peptide in contrast to other peptides that required ERAAP.

In this study, we examine how the presentation of the SVL9 peptide is negatively regulated by ERAAP. With insights from the Db binding characteristics of the SVL9 peptide and the crystal structure of the SVL9-Db complex (16), we analyzed the generation of the SVL9 peptide in the presence or absence of other Db binding peptides. We find that relative levels of SVL9 presentation are determined not only by its ability to bind Db but also on the presence of competitor peptides and ERAAP in the ER.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Cell lines and DNA constructs

The β-galactosidase (lacZ)-inducible SVL9/Db-specific 30NXZ, WI9/Db-specific 11P9Z, LYL8/Kb-specific BCZ103, and SHL8/Kb-specific B3Z hybridomas, the TAP plus ERAAP double-deficient fibroblasts as well as TAP-deficient fibroblasts have been described earlier (10, 14, 17, 18). The immortalized KbDb and ERAAP-deficient fibroblasts were established as described (19). The ER-targeted ER-translocation signal (ES)-X5[SVL9] (ES-AMQLK[SSVVGVWYL]), ES[SVL9] (ES[SSVVGVWYL]), ES-X7[WI9] (ES-AIVMQLK[WMHHNMDLI]), ES[WI9] (ES[WMHHNMDLI]), ES-X7[LYL8] (ES-AIVMQLK[LTFNYRNL]), and ES-X5[SHL8] (ES-AIVMK[SIINFEHL]) were subcloned into pcDNA1 vector (20). The ES-X5[SVNL9] (ES-AMQLK[SSVVNVWYL]) construct was made by site directed mutagenesis beginning with the ES-X5[SVL9] construct using a complementary primer pair (5' CAG CTT AAG TCC TCC GTG GTG AAC GTG TGG TAC CTG TAG TAG 3' and 5' CTA CTA CAG GTA CCA CAC GTT CAC CAC GGA GGA CTT AAG CTG 3').

T cell activation assays

For transient transfections, the DNA constructs were introduced into the indicated recipient cells by FuGENE6 (Roche) according to the manufacturer’s instruction and assayed 2 days later. For transfections with multiple cDNAs, the relative proportion of different constructs and the total DNA amount (e.g., 3 µg x 3 = 9 µg in total for triple DNA transfection) was held constant in each experiment. The reproducibility of the data was established in independent experiments. The transfected cells were titrated and incubated overnight with the indicated T cell hybridomas. The lacZ activity induced upon T cell activation was measured by the conversion of the substrate chlorophenolred-β-D-galactopyrannoside to chlorophenol red by its absorbance at 595 nm with 655 nm as the reference wavelength (21). Alternatively, peptide extracts of transfected cells were prepared by acid extraction, dried, and assayed for antigenic activities by incubation with indicated T cell hybridomas using either Kb or Db expressing L cells as APC. In Fig. 3, the cells were treated with 8 µg/ml Brefeldin A (BfA) or medium alone for 2 or 4 h, then cocultured with the T cell hybridomas in the presence of BfA to prevent further pMHC I expression during culture.

Reversed phase (RP)-HPLC fractionation of the peptide and precursor peptides

The preparation of the peptide extracts and analysis by RP-HPLC fractionation has been described (20). Briefly, the extracts from 2 x 106 transfected cells with indicated DNA constructs were filtered through Microcon YM-10 m.w. cut-off filters (Amicon). The <10kD filtrate was fractionated by HPLC on a RP C18 column (Vydac). Fractionated samples were dried by vacuum centrifugation. All the ES-Xn[peptide] constructs used here contain a lysine residue flanking the N terminus of the final antigenic peptide, which allows release of the optimally active peptide after trypsin (50 µg/ml) treatment (22). After coculture with the indicated T cell hybridomas and APCs expressing appropriate MHCI, the induced lacZ activity was measured as above. The elution profiles of SHL8, K[SHL8], SVL9, or WI9 peptides were determined by running synthetic peptides under identical conditions. Mock runs with sample buffer only were run and assayed under identical conditions to ensure absence of cross-contamination between samples.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Generation of processed SVL9/Db is inhibited by Db binding peptide in the ER

Previous analysis has shown that unlike many peptides that were no longer presented in ERAAP-deficient cells, those derived from the H13 and the H47 histocompatibility genes were significantly over-expressed (10, 11). These peptides were, therefore, classified as ERAAP-sensitive peptides (9). We noticed that the H13 encoded peptide SVL9 (SSVVGVWYL), as well as the H47 peptide (SCILLYIVI), did not conform to the consensus motif (XXXX[N]XXX[I,M,L]) for peptides presented by the Db MHC (23). Although both peptides are nine residues long, they lack the asparagine residue at p5, suggesting that their MHC binding characteristics may differ from other Db canonical peptides, such as the WI9 (WMHHNMDLI) or the K[SHL8] (KSIINFEHL) peptides. Because, as a typical MHC molecule, Db simultaneously presents a large mixture of peptides, we asked whether the presence of other Db binding peptides influenced presentation of the ERAAP-sensitive SVL9/Db complex.

We first used precursors encoding the OVA peptide, SIINFEHL (SHL8) presented by the Kb, and the overlapping peptide, KSIINFEHL (K[SHL8]) presented by Db MHC (24). In HPLC fractionated peptide extracts of KbDb-deficient fibroblasts cotransfected with the ER-targeted, ES-X5[SHL8] construct and either Kb or Db cDNAs, only the processed SHL8 or the K[SHL8] peptide was detected and there was no discernible activity in vector transfected cells (Fig. 1A). Thus, processing of the same precursor in the ER yields two different peptide products strictly defined by the presence of Kb or Db MHC.


Figure 1
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  		FIGURE 1. Processing of ER-targeted ES-X5[SHL8] precursor yields peptides presented by Kb and Db and inhibits the generation of the SVL9 peptide in the ER. A, The (KbDb-ERAAP)-deficient B6 fibroblasts were cotransfected with ES-X5[SHL8] (ES-AIVMK[SIINFEHL]) and full-length ERAAP, together with either Kb or Db MHC class I cDNAs. ES-X5[SHL8] construct contained the ER-targeted signal sequence, ES, upstream of the precursor containing five N-terminal flanking residues and the SHL8 octapeptide. The cell extracts (<10kDa) of transfected cells were fractionated by HPLC, and each fraction was treated with trypsin to release the SHL8 peptide from the N-terminally extended peptides and assayed with SHL8/Kb-specific B3Z hybridoma and Kb-L cells as APC. After overnight incubation, the lacZ activity induced in the hybridoma was measured with the substrate chlorophenolred-β-D-galactopyrannoside whose product absorbs light at 595 nm. Fractions collected from the mock HPLC run with a buffer alone were assayed in parallel to test for cross-contamination between runs. B, The TAP-deficient B6 fibroblasts expressing Kb and Db were cotransfected with ES-X5[SVL9] and ES-X5[SHL8], or vector. Two days after transfection, the <10kD extracts of transfected cells were titrated and SVL9 peptide activity was measured as above using the SVL9/Db-specific 30NXZ T cells and Db-L cells as APC. Data are representative of two independent experiments.

 
To determine whether the presence of other MHC binding peptides influenced the processing of the SVL9 peptide, we used TAP-deficient fibroblasts as APCs. In these cells, the absence of TAP prevents cytosolic peptides from entering the ER (4), making it possible to confine the processing and presentation of antigenic precursors in the ER compartment (20, 25, 26). Precursors were targeted into the ER by appending an ES upstream of the antigenic peptide flanked by five amino acids (X5 = AMQLK) (20). The SVL9 precursor ES-X5[SVL9] DNA was cotransfected with the ES-X5[SHL8] construct or the vector. Two days later, the peptides were extracted from the transfected cells and the generation of the SVL9 peptide was measured with the 30NXZ hybridoma and Db-L cells as APCs (14) (Fig. 1B). Compared with vector transfected cells, the amount of SVL9 peptide in the extracts was substantially lower in cells cotransfected with the ES-X5[SHL8] construct. We infer that processing of the SVL9 peptide in the ER was influenced by the presence of other peptides processed by the same cells.

The ES-X5[SHL8] precursor yields both Kb and Db binding SHL8 and K[SHL8] peptides, respectively (Fig. 1A). To determine whether one or both pMHC I played a role in inhibiting the presentation of SVL9, we transfected TAP-deficient cells with the Ag construct ES-X5[SVL9], together with equal amounts of ES-X7[WI9] construct DNA. The WI9 peptide is derived from the Y-chromosome encoded Uty histocompatibility genes and is only presented by the Db MHC (27). As above, ER proteolysis efficiently trimmed the N-terminal flanking residues from the SVL9 peptide to generate the final SVL9/Db complex when cells were cotransfected with vector alone (Fig. 2A). However, the T cell response to the SVL9/Db complex was lower when the cells also expressed the ES-X7[WI9] construct encoding the WI9 peptide. Because the T cell response to pMHC I on the cell surface is not quantitative, we measured the amount of processed SVL9 peptide extracted from transfected cells in an exogenous presentation assay using Db-L cells as APC (Fig. 2B). The relative amount of SVL9 peptide in extracts of cells with vector alone vs cells expressing WI9 peptide was reduced by over 90%. Thus, the presence of WI9 peptide was deleterious to the presentation of the SVL9/Db complex.


Figure 2
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  		FIGURE 2. The generation and presentation of processed SVL9/Db complex is inhibited by the Db but not Kb binding peptides in the ER. The TAP-deficient B6 fibroblasts expressing Kb and Db MHC were cotransfected with the indicated cDNAs encoding ER-targeted antigenic precursors together with potential competitor constructs. After 2 days, indicated numbers of transfected cells were cocultured either with (A) SVL9/Db-specific 30NXZ hybridoma or (C) WI9/Db-specific 11P9Z hybridoma. After overnight incubation, the lacZ activity induced in the hybridomas was measured as above. B and D, The peptide extracts of cells transfected with the indicated cDNA constructs, 2 days earlier, were titrated using Db-L cells as APC. The presence of the SVL9 or the WI9 peptides was assayed by the 30NXZ or the 11P9Z T cell hybridomas as described above. E and F, TAP-deficient fibroblasts were transfected with the indicated cDNA constructs and varying numbers of cells were used as APCs for (E) the SVL9/Db-specific 30NXZ, or (F) the LYL8/Kb-specific BCZ103 hybridoma. Data are representative of two (five for B) independent experiments.

 
The inhibition of processed SVL9 in the presence of the WI9 peptide was unidirectional and specific for the Db MHC. In a reciprocal experiment, we used the 11P9Z T cell hybrid to measure the generation of the WI9/Db complex on the surface, or in extracts, of cells transfected with the ER-targeted ES-X7[WI9] construct (Fig. 2, C and D). The generation of the WI9/Db complex on the cell surface or the naturally processed WI9 peptide in the extract was not affected by the presence of the SVL9 peptide. Likewise, the presentation of the SVL9/Db complex was not affected by cotransfecting cells with an equal amount of ES-X7[LYL8] construct DNA. The ES-X7[LYL8] construct encodes an unrelated LYL8 (LTFNYRNL) peptide (18), which is presented by the Kb MHC in the same cells (Fig. 2E). Conversely, the presentation of the LYL8/Kb complex detected by the BCZ103 hybridoma was also not affected by coexpression of SVL9 peptide presented by Db MHC (Fig. 2F). We conclude that the generation of the processed SVL9 peptide and its presentation as the SVL9/Db complex was inhibited only by other Db binding peptides.

Stability of SVL9-Db complex is enhanced by substituting the p5 anchor residue

The naturally processed SVL9 nonapeptide, SSVVGWVYL, without asparagine at the p5 position does not conform to the XXXX[N]XXX[I,L,M] consensus motif for Db binding peptides (23). Analysis of the crystal structure of the SVL9 peptide bound to the Db MHC showed that the C pocket in Db, which is normally occupied by the side chain of the p5 asparagine anchor, is instead occupied by the smaller p5 glycine residue and water molecules (16). In this study, substituting the asparagine for the glycine residue in the p5 position of the SVL9 sequence (SVNL9, SSVVNWVYL), was shown to increase its Db binding affinity ~10-fold. Furthermore, structural analysis of Db bound to either SVL9 vs SVNL9 peptides showed that the increase in affinity correlated with displacement of water molecules in the C-pocket of the peptide-Db crystals.

We assessed whether enhancing the affinity of the SVL9 peptide by substitution of the p5 glycine residue by asparagine (G5N) would alter its presentation by Db on the cell surface. COS cells were transfected with the ES-X5[SVL9] or the ES-X5[SVNL9] construct with either Db or Kb cDNAs. The 30NXZ T hybridoma responded to cells expressing Db MHC and either construct encoding the antigenic SVL9 or SVNL9 peptides (Fig. 3, A and B). Cells cotransfected with the Ag constructs and Kb did not activate the 30NXZ T cell. Thus, the 30NXZ response to the G5N substituted SVNL9 peptide was clearly specific and Db restricted although it was reproducibly lower than that of the wild-type SVL9 peptide. Presumably, subtle structural differences between the SVNL9-Db and the wild-type SVL9-Db complexes can be distinguished by the TCR of the 30NXZ hybridoma.


Figure 3
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  		FIGURE 3. Substituting the p5 glycine with asparagine residue in SVL9 peptide improves stability of the peptide/Db complex. The COS cells were cotransfected with either (A) ES-X5[SVL9] or (B) ES-X5[SVNL9] constructs with the cDNAs encoding Db or Kb MHC. Two days later, the lacZ response of the 30NXZ hybridoma was determined after coincubation with the indicated number of transfected cells. The TAP-deficient fibroblasts were transfected either with (C) vector alone, (D) ES-X7[WI9], (E) ES-X5[SVL9], or (F) ES-X5[SVNL9]. After 2 days, the cells were treated with BfA for 2 or 4 h to block expression of newly assembled pMHC I. The remaining pMHC I complexes on the surface were measured by indicated hybridoma response. Data are representative of two independent experiments.

 
Next, we tested whether the G5N substitution increased the stability of the SVL9 analogues when presented by Db. TAP-deficient fibroblasts were transfected with cDNA constructs encoding ER-targeted SVL9, SVNL9, or the WI9 peptide as a positive control. After 2 days, the transfectants were incubated for varying time periods with BfA, a drug that blocks ER to Golgi traffic and thus inhibits the transport of newly assembled pMHC I to the cell surface (28). The presence of pMHC I on the surface of BfA-treated cells was measured with either the WI9/Db or SVL9 or SVNL9/Db-specific 30NXZ T cells (Fig. 3, C–F). There was no detectable difference in the surface expression of the WI9/Db complex after either 2 or 4 h incubation with BfA. In contrast, consistent with its weak Db binding affinity, the expression of the peptide SVL9-Db complex on the cell surface decreased after 4 h of BfA treatment. The loss of pMHC I ligand upon BfA treatment was, however, not observed in cells transfected with the ES-X5[SVNL9] construct. We conclude that the G5N substitution in the p5 anchor position of the SVL9 sequence enhanced the stability of the peptide-Db complex not only in vitro (16) but also in vivo.

The presentation of substituted SVNL9 peptide is less affected by competitor peptide

We determined whether the increase in the stability of SVNL9 bound Db complex influenced the ability of the WI9 peptide to inhibit its generation in the ER. We cotransfected TAP-deficient cells with the Ag coding ES-X5[SVNL9] precursor together with either vector or the ES-X7[WI9] constructs. In contrast to the profound inhibition in the presentation of the SVL9 peptide (Fig. 2B), the WI9 peptide had a relatively smaller effect on the generation of the SVNL9 peptide either on the cell surface as pMHC I or in cell extracts (Fig. 4, A and B). Again there was no measurable effect of the SVNL9 peptide on the presentation of the WI9 peptide on the cell surface or in cell extracts, suggesting that even with the p5 asparagine residue SVNL9 peptide did not outcompete the WI9 peptide (Fig. 4, C and D). Quantitative comparison of the peptide amounts in cell extracts showed that the presence of the WI9 peptide inhibited the recovery of the wild-type SVL9 peptide by ~90% but inhibited the SVNL9 peptide by only 50%. (Fig. 4, E and F). Thus, presentation of the SVL9 analogues in presence of the competitor WI9 peptide was proportional to their stability as Db complexes.


Figure 4
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  		FIGURE 4. The presentation of the SVNL9 mutant peptide is less susceptible to inhibition by the WI9 peptide. The TAP-deficient B6 fibroblasts were cotransfected with the indicated cDNAs encoding ES-X5[SVNL9] antigenic precursor together with the competitor ES-X7[WI9] construct. After 2 days, indicated numbers of transfected cells were cocultured either with (A) SVL9/Db-specific 30NXZ hybridoma or (C) WI9/Db-specific 11P9Z hybridoma and the lacZ activity measured as above. B and D, The peptide extracts of cells transfected with the indicated cDNA constructs, 2 days earlier, were titrated using Db-L cells as APC. The presence of the SVL9 or the WI9 peptides was assayed by the 30NXZ or the 11P9Z T cell hybridomas as described above. E and F, The percent peptide recovery of the SVL9 or the SVNL9 peptides in the absence or presence of the competitor WI9 peptide based upon the 30NXZ T cell responses in Figs. 2B and 6B. Data are representative of two (four for B) independent experiments (A–D) or calculated from results of three independent experiments (E and F; *, p < 0.05).

 
ERAAP is required for generation as well as destruction of the SVL9 peptide

The enzymatic activity of ERAAP is essential for the generation of the final pMHC I from precursors containing N-terminal flanking residues in the ER compartment (10, 26). To determine whether ERAAP was involved in regulating competitive peptide presentation by MHC I molecules, we first tested the requirement for ERAAP in the generation of the final peptides from their N-terminally extended precursors. We transfected (TAP+ERAAP)-deficient fibroblasts with constructs encoding the ER-targeted, N-terminally extended ES-X5[SVL9] and ES-X5[WI9] precursors. The presentation of the SVL9 or the WI9 peptides by Db was measured with 30NXZ and 11P9Z T cell hybridomas (Fig. 5, A and B). The SVL9-Db, as well as the WI9-Db complexes, was efficiently expressed on the cell surface only when the cells were cotransfected with the full-length ERAAP cDNA but not with vector alone. Thus, ERAAP was essential for trimming the N-terminal flanking residues in the SVL9 as well as the WI9 precursors to generate the final pMHC I.


Figure 5
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  		FIGURE 5. The generation as well as destruction of processed SVL9 peptide requires ERAAP activity. The (TAP+ERAAP)-deficient fibroblasts were cotransfected with ES-X5[SVL9] and ES-X7[WI9], with or without the full-length ERAAP cDNA construct. Then antigenic peptide/MHC complex expressed on the surface was measured by the lacZ response of either (A) the SVL9/Db-specific 30NXZ or (B) the WI9/Db-specific 11P9Z hybridoma. C and D, The cell extracts from the (TAP+ERAAP)-deficient fibroblasts cotransfected with ES-X5[SVL9] without (C) or with (D) the competitor ES-X7[WI9] and full-length ERAAP or vector cDNA were fractionated by HPLC. Each fraction was treated with trypsin and assayed for SVL9 antigenic activity with 30NXZ hybridoma and Db-L cells as APC. The arrows indicate the peak antigenic activity of the SVL9 peptide or its potential precursor. Data are representative of two independent experiments.

 
To directly examine the proteolysis of antigenic precursors in the ER, we prepared a peptide extract from transfected (TAP+ERAAP)-deficient fibroblasts and fractionated it by HPLC. As in other precursors used earlier (26), the SVL9 peptide in the ES-X5[SVL9] precursor is preceded by a lysine residue that allows trypsin to cleave the C terminus of lysine and release the optimally active SVL9 peptide from its poorly antigenic N-terminally extended precursor (22, 26). Each HPLC fraction was treated with trypsin before assaying for SVL9 activity with 30NXZ hybridoma and Db-L cells as APCs. In cells transfected with the ES-X5[SVL9] construct and ERAAP, a single peak of antigenic activity (fraction no. 34) was detected, which coincides with the synthetic SVL9 peptide run under identical conditions (Fig. 5C). In contrast, the SVL9 peptide was not detected in HPLC fractionated peptide extract of the same cells without ERAAP. Instead the antigenic activity was present in a later HPLC fraction no. 40, which is most likely the N-terminally extended precursor of SVL9 peptide. In the presence of ERAAP, the amount of the final SVL9 peptide was comparable to that of the antigenic precursor detected in the absence of ERAAP. This result directly confirmed that the N-terminally extended precursor of SVL9 peptide was efficiently converted by ERAAP to the precise SVL9 peptide product in the ER.

To determine how the presence of the WI9 peptide affected the processing of the SVL9 peptide, we analyzed extracts of (TAP+ERAAP)-deficient cells transfected with the ES-X5[SVL9] as well as the competitor ES-X7[WI9] in the presence or absence of ERAAP (Fig. 5D). Again, in cells expressing the SVL9-precursor without ERAAP, only the precursor peptide was found in fractions no. 39–40 and the processed SVL9 peptide was not detected. In contrast, extracts of cells cotransfected with ERAAP did contain the SVL9 peptide in fraction no. 34 but its recovery was substantially lower compared with that of the precursor peptide. Thus, although ERAAP was required for trimming the SVL9 precursor with or without the WI9 competitor, ERAAP also inhibited the generation of SVL9 product in the presence of the Db binding WI9 peptide.

ERAAP limits presentation of SVL9 without precursor trimming

Finally, we asked whether the inhibition of the SVL9-Db complex caused by the WI9 peptide was due to competition for binding a limiting number of peptide receptive Db and/or because ERAAP was required to trim the antigenic precursor. To directly examine the presentation of the final peptides in the absence of ERAAP, we used precursor constructs without any N-terminal flanking residues between the ES signal sequence and the antigenic peptides. The (TAP+ERAAP)-deficient cells were cotransfected with either the ES-[SVL9] or the ES-[WI9] constructs, and their peptide extracts were fractionated by HPLC (Fig. 6, A and B). Each fraction was tested for the presence of antigenic activity using SVL9/Db or the WI9/Db-specific hybridomas. In HPLC fractionated extracts, only a single peak of activity was detected which co-eluted with the synthetic SVL9 (fraction no. 34–35) or the WI9 peptide (fraction no. 13–14). Thus, as seen before with the SHL8-Kb presentation (10, 20), ERAAP was not required for generating the processed SVL9 and the WI9 peptides when the antigenic precursors in the ER do not contain any N-terminal flanking residues.


Figure 6
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  		FIGURE 6. The ERAAP inhibits SVL9 presentation in presence of Db and without need to trim the SVL9 precursor. (A and B) Peptides extracted from (TAP+ERAAP)-deficient fibroblasts transfected with ES-[SVL9] or ES-[WI9] constructs were fractionated by HPLC. Each fraction was assayed for antigenic activity for stimulating the 30NXZ or 11P9Z hybridomas using Db-L cells as APCs. Fractions collected without sample (mock) were also assayed in parallel to rule out contamination between runs are shown as a line. C–F, The (TAP+ERAAP)-deficient fibroblasts were cotransfected with ES-[SVL9] together with the ES-[WI9] or vector constructs in the (C and D) absence or (E and F) presence of ERAAP. The cells were assayed as APC for stimulating the 30NXZ hybridoma as above. G, The (TAP+ERAAP)-deficient fibroblasts were cotransfected with ES-[SVL9] together with the ES-[WI9] in the absence or presence of ERAAP. Peptides extracted from the cells were fractionated by HPLC and assayed by 30NXZ as described above. Data are representative of two independent experiments.

 
To assess the ability of the WI9 peptide to inhibit the SVL9 presentation, we cotransfected the ES-[SVL9] with either the ES-[WI9] construct or the vector into (TAP+ERAAP)-deficient fibroblasts. The transfected cells were used as APCs or the processed peptides were analyzed in cell extracts by their ability to stimulate the SVL9-Db-specific 30NXZ hybridoma. In the absence of ERAAP, the WI9 peptide had little if any effect on the generation of the SVL9-Db complex on the cell surface (Fig. 6C) or in cell extracts (Fig. 6D). Thus, the mere presence of WI9 peptide did not affect the presentation of the SVL9-Db complex, demonstrating that the amount of peptide receptive Db was not limiting. In contrast, when the ERAAP cDNA was also transfected into the cells, the WI9 peptide inhibited presentation of SVL9-Db on the cell surface as well as the processed SVL9 peptide in cell extracts (Fig. 6, E and F). To further confirm this result, we compared the SVL9 peptide recovered in cell extracts by HPLC fractionation (Fig. 6G). A single peak of antigenic activity corresponding to the synthetic SVL9 peptide was observed in the absence of ERAAP. However, this activity was largely absent when ERAAP was cotransfected in the cells. Thus, inhibition of the SVL9 peptide in presence of the WI9 peptide could be attributed to ERAAP function.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
The repertoire of diverse peptides presented by MHC I molecules on the cell surface, and available for recognition by CD8 T cell repertoire, is shaped by the aminopeptidase ERAAP in the ER. This is evident from the selective disruption of the normally diverse pMHC I repertoire in ERAAP-deficient vs wild-type cells (9, 10, 11). Although some pMHC I remain unaffected, a significant fraction of normal pMHC I, such as the WI9 peptide encoded by the Y-chromosome, Uty histocompatibility gene, is lost from the surface of ERAAP-deficient spleen cells (10, 11). Notably, in the same ERAAP-deficient cells, expression of other sets of peptides increases dramatically. This category includes yet uncharacterized but presumably "unedited" peptides as well as those encoded by the H13 and H47 histocompatibility genes. Thus, all pMHC I are not equal; the expression of pMHC I is qualitatively and quantitatively regulated by ERAAP.

The mechanism by which ERAAP regulates presentation of individual peptides is intriguing. We showed earlier that in vivo the ability of ERAAP to trim antigenic precursors into their final form requires MHC I molecules (26). In the absence of appropriate MHC I, ERAAP only degraded the antigenic precursors in the ER. In contrast to the notion that ERAAP acts independently as a "molecular ruler" (6, 29, 30), in this view ERAAP uses the MHC I molecule as a template for trimming peptides that are eventually presented by MHC I. Thus, it was unexpected that ERAAP was also involved in markedly reducing the presentation of another set of peptides.

Earlier measurements from our laboratory and independently by the van Kaer group have shown that the SVL9/Db complex is consistently and significantly up-regulated in ERAAP-deficient cell lines or normal spleen cells (5, 10, 11, 20). According to the models discussed above, SVL9 could have been independently destroyed due to the intrinsic substrate specificity of ERAAP itself (6, 30). Alternatively, the destruction of SVL9 by ERAAP could have been influenced by MHC because other Db binding peptides are present in wild-type cells but are missing in the absence of ERAAP. Our results show that the susceptibility of the noncanonical SVL9 to destruction by ERAAP is influenced by competition with other Db binding peptides. Furthermore, the inhibition was not due to limiting availability of Db MHC or ERAAP, because it was also observed with precursors that did not require ERAAP trimming for presentation. Thus, the presentation of the SVL9 peptide was influenced by both ERAAP and Db MHC.

The susceptibility of the SVL9 peptide to destruction by ERAAP correlates inversely with the ability of this peptide to bind Db. This is remarkably consistent with previous results showing that the SVL9 peptide was bound weakly by Db and binding could be improved by replacing the p5 glycine in SVL9 with the canonical p5 asparagine anchor residue (16). Indeed substituting the p5 glycine with the asparagine residue in the SVNL9 peptide improved both the Db binding and reduced the susceptibility to destruction by ERAAP.

The simplest model to explain the role of ERAAP in regulating the diverse pMHC I display is to postulate that the precursor peptides available for MHC I loading in the ER differ in their susceptibility to ERAAP (6, 29, 30, 31). Accordingly, the increase in SVL9 presentation in ERAAP-deficient cells would be due to susceptibility of SVL9 to destruction by ERAAP in wild-type cells as well to competition for MHC I binding by other peptides. The ERAAP-mediated destruction of SVL9 may be further exacerbated by the poorer Db binding capacity relative to other peptides. Alternatively, because ERAAP has been shown to destroy precursor peptides when they are not bound by MHC I (26), it is possible that that ERAAP may function not before but after the peptides have been tested for binding to MHC I in the ER. Peptides that bind sufficiently well to the MHC I, such as the WI9 peptide, exit the ER, while those such as the SVL9 peptide might linger longer in the ER and become susceptible to destruction by ERAAP. The latter model appears counterintuitive but is consistent with the idea that ERAAP trims precursors while they are bound to MHC I (26, 32, 33). Furthermore, escape from the ER of some of these MHC I with weakly bound peptides could explain why ERAAP-deficient cells appear immunogenic to otherwise identical wild-type mice (9).

In conclusion, our findings suggest that ERAAP functions in regulating the quality of processed peptides suitable for presentation. In concert with MHC I molecules, ERAAP can also destroy unstable peptides and thus not only enhances but also limits pMHC I presentation. It should be interesting to determine whether dramatic changes in the presentation of endogenous peptides of the H13 histocompatibility gene influence its established role in rejection of tissue grafts (34, 35). Likewise some viral epitopes have also been observed to be up-regulated in ERAAP-deficient cells (11, 12, 13), but their role in anti-viral immunity remains to be elucidated (36).


   Acknowledgments
 
We acknowledge Gianna Hammer for providing the fibroblast cell lines of various genotypes.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by grants from the National Institutes of Health (to N.S.). T.K. is a research fellow of the Japan Society for the Promotion of Science. Back

2 Address correspondence and reprint requests to Dr. Nilabh Shastri, Division of Immunology, LSA 421, Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720-3200. E-mail address: nshastri{at}berkeley.edu Back

3 Abbreviations used in this paper: pMHC I, peptide presented by MHC class I; ER, endoplasmic reticulum; ERAAP, ER aminopeptidase associated with Ag processing; RP, reversed phase; BfA, Brefeldin A; ES, ER-translocation signal. Back

Received for publication June 27, 2008. Accepted for publication August 25, 2008.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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