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Originally published In Press as doi:10.1074/jbc.M105974200 on July 30, 2001

J. Biol. Chem., Vol. 276, Issue 41, 38255-38260, October 12, 2001
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Pleiotropic Effects of Post-translational Modifications on the Fate of Viral Glycopeptides as Cytotoxic T Cell Epitopes*

Denis HudrisierDagger §, Joëlle RiondDagger , Honoré MazarguilDagger , and Jean Edouard GairinDagger ||

From the Dagger  Institut de Pharmacologie et de Biologie Structurale, UMR5089 CNRS/Université Paul Sabatier, 205 route de Narbonne, 31400 Toulouse, France and the § Institut National de la Santé et de la Recherche Médicale, U395, CHU Purpan, BP3028, 31024 Toulouse Cedex 03, France

Received for publication, June 27, 2001, and in revised form, July 26, 2001


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The fate of viral glycopeptides as cytotoxic T lymphocyte (CTL) epitopes is unclear. We have dissected the mechanisms of antigen presentation and CTL recognition of the peptide GP392-400 (WLVTNGSYL) from the lymphocytic choriomeningitis virus (LCMV) and compared them with those of the previously reported GP92-101 antigen (CSANNSHHYI). Both GP392-400 and GP92-101 bear a glycosylation motif, are naturally N-glycosylated in the mature viral glycoproteins, bind to major histocompatibility complex H-2Db molecules, and are immunogenic. However, post-translational modifications differentially affected GP92-101 and GP392-400. Upon N-glycosylation or de-N-glycosylation, a marked decrease in major histocompatibility complex binding was observed for GP392-400 but not for GP92-101. Further, under its N-glycosylated or de-N-glycosylated form, GP392-400 then lost its initial ability to generate a CTL response in mice, whereas GP92-101 was still immunogenic under the same conditions. The genetically encoded form of GP392-400, which on the basis of its immunogenicity could still be presented with H-2Db during the course of LCMV infection, does not in fact appear at the surface of LCMV-infected cells. Our results show that post-translational modifications of viral glycopeptides can have pleiotropic effects on their presentation to and recognition by CTL that contribute to either creation of neo-epitopes or destruction of potential epitopes.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Viral peptides presented by major histocompatibility complex (MHC)1 class I molecules at the surface of infected cells to CTL are key molecular signals allowing recognition of the infected cells and their subsequent destruction by the activated CTL. Those peptides commonly consist of octameric to undecameric antigenic peptides generated from viral proteins by a multistep, intracellular processing pathway involving components present in the cytoplasm and the endoplasmic reticulum. Peptides generated in the cytosol from the degradation of ubiquitinated proteins by the proteasome and possibly by nonproteasomal cytosolic proteases (1, 2) are then translocated via peptide-specific transporters (transporter associated with antigen processing) into the endoplasmic reticulum where they assemble with the MHC class I molecules and are eventually subjected to further trimming (3, 4). The capacity or contrastingly the inability of a given peptide to proceed through these steps has been shown to determine its presentation by MHC molecules (5-7). In contrast to the well studied processing pathway of peptides derived from cytosolic proteins, very little is known about the ability of glycopeptides originating from viral glycoproteins to successfully pass through the above mentioned steps. For example based on the use of model peptides chemically glycosylated (8-12) or affected by other post-translational modifications such as reduction of sulfhydryl groups (13), bond rearrangement (14), or phosphorylation (15), it has been proposed that these modifications could generate new epitopes and thus increase antigenic diversity (10, 11, 16-19). However, given the failure to identify naturally processed N-glycopeptides as epitopes to date, the fate of viral glycopeptides as MHC class I-restricted antigens remains very unclear.

Lymphocytic choriomeningitis virus (LCMV) infection of H-2b mice is cleared by CTL that recognize immunodominant (NP396-404, GP33-41/43, and GP376-386) (20-24) or subdominant (GP92-101) epitopes presented in the context of a MHC class I H-2Db and/or H-2Kb molecule (16, 20, 25, 26). The presence of glycopeptides derived from the LCMV glycoproteins bearing the H-2Db binding motif makes LCMV infection a model of choice to study the fate of glycopeptides as CTL epitopes. We recently showed that the LCMV GP92-101 subdominant epitope (CSANNSHHYI), which bears the glycosylation motif NNS within its sequence, was post-translationally modified in infected cells and presented at the cell surface by H-2Db molecules in two different forms, a nonglycosylated form (CSANNSHHYI) and a de-N-glycosylated form (CSADNSHHYI) resulting from the N-glycanase-mediated de-N-glycosylation of a previously N-glycosylated peptide sequence (16).

An additional H-2Db-restricted glycopeptide GP392-400 (WLVTNGSYL) was previously identified in the LCMV GP2 protein (25). It shares similar biochemical properties with GP92-101. Like GP92-101, GP392-400: (i) bears a NXS glycosylation motif, (ii) is N-glycosylated in the mature LCMV GP2 glycoprotein (27), and (iii) binds efficiently to H-2Db (25, 28). In contrast to GP92-101, no CTL response against GP392-400 has been detected (28), and its cellular status remains unknown. The contrasting immunogenic properties of GP92-101 and GP392-400 suggest that the common biochemical events or intracellular mechanisms encountered by these two viral glycopeptides along the processing and presentation pathway lead to different fates as MHC class I-restricted CTL epitopes. This would not have been predicted on the basis of their biochemical properties. The aim of our study was therefore to explore and dissect the intracellular molecular mechanisms that may differentially affect the processing pathways of these two viral glycopeptides and explain their different antigenic properties.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

CTL, Cell Lines, and Virus-- CTL were obtained from C57BL/6 mice immunized subcutaneously at the base of the tail with 50-100 µg of the indicated peptides mixed with 5 µg of P30 T-helper epitope from tetanus toxoid in incomplete Freund's adjuvant. After 1 week, draining lymph nodes were removed, and CTL were restimulated at weekly intervals with irradiated (2,500 rads) C57BL/6 splenocytes and irradiated (10,000 rads) peptide-pulsed (1 µM) RMA cells in the presence of 30 IU/ml interleukin-2 (EL4 supernatant). Murine H-2b mutant RMA-S cells were used in binding experiments. The murine H-2b cell lines RMA and MC57 were used in in vitro cytotoxicity assays. The cells were grown in Dulbecco's modified Eagle's medium (RMA, RMA-S, and MC57) containing 5% bovine serum, L-glutamine (2 mM), and antibiotics (10 units/ml penicillin and 10 µg/ml streptomycin). The LCMV Armstrong strain (LCMV Arm) was used to infect mice or cells.

Peptide Synthesis-- The peptides were synthesized by the solid phase method using Fmoc chemistry. For Asn-glycosylated peptides, Fmoc-Asn(Ac3AcNH-beta Glc)-OH, a commercially available derivative of Asn bearing GlcNAc (Novabiochem) was used. After standard protocols for solid phase synthesis, cleavage, and deprotection, the glycopeptide was de-O-acetylated with 0.1 M sodium hydroxide as described previously (16). The peptides were purified by HPLC on a RP300-C8 reversed phase column (Brownlee Lab), and their identities were confirmed by electrospray ionization mass spectrometry.

Cytotoxicity Assays-- RMA or MC57 cells were incubated for 1 h at 37 °C in medium containing 10-fold dilutions of peptides. MC57 cells were infected at a multiplicity of infection of 2 with LCMV Arm 48 h prior to the assay. RMA (peptide-pulsed or not) or MC57 (LCMV-infected or not) cells were 51Cr-labeled and used as targets (5 × 103/well) in chromium release assays. CTL (1.5 × 104/well or at the indicated E/T ratio) were added, and after 4 h of incubation at 37 °C, the 51Cr content of supernatants was determined. The specific lysis was calculated as 100 × [(experimental - spontaneous release)/(total - spontaneous release)].

Binding Studies-- The binding studies were performed as described previously (25, 29). Briefly, RMA-S cells (5 × 105 cells/well) previously incubated at 26 °C for 40 h were placed in U-bottomed 96-well plates for 4 h at 37 °C with increasing concentrations (0-10-4 M) of unlabeled peptides. The cells were then washed twice with BSA/PBS and incubated at 4 °C for 45 min. with the 28-14-8S anti-H-2Db monoclonal antibody (hybridoma supernatant) (30). After two washes in BSA/PBS, an anti-mouse IgG antibody conjugated to fluorescein isothiocyanate (Sigma) was added for 45 min. Then the cells were washed twice in BSA/PBS and analyzed by flow cytometry.

Peptide Extraction from LCMV-infected Cells-- The peptides were extracted from the surface of LCMV-infected MC57 (H-2b) cells as described previously (16). Briefly, cells (1-2 × 109) were washed in PBS and then resuspended in 0.1 M citrate/phosphate buffer at pH 3.0 for 2 min. The eluted material was desalted on a SepPak column, transferred onto a Centricon 10, and centrifuged at 3500 rpm for 30 min at 4 °C. Material less than 10 kDa was vacuum concentrated then resuspended in 20 µl of 0.08% trifluoroacetic acid. The peptides were separated by HPLC (Waters 600S controller system) on a reversed phase C18 column (7 µm, 2.1 × 100 mm, Aquapore, BrownleeTM) according to the following procedure: solution A, 0.08% trifluoroacetic acid in H2O; solution B, 0.08% trifluoroacetic acid in CH3CN; a 60-min gradient, 5-60% B; flow rate: 400 µl/min. The fractions (200 µl) were collected in U-bottomed 96-well plates, lyophilized, reconstituted in PBS, and stored at -80 °C before analysis.

Molecular Modeling-- The three-dimensional models for LCMV GP92-101 (CSANNSHHYI) and LCMV GP392-400 (WLVTNGSYL) interacting with H-2Db have been produced using the online modeling facility SwissModel (GlaxoWellcome) at the following address: www.expasy.ch/spdbv/.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The Genetically Encoded Form of LCMV GP392-400 but Neither Its N-Glycosylated nor De-N-glycosylated Forms Can Generate a CTL Response in H-2b Mice-- Peptides bearing a N-glycosylation motif NX(S/T) can theoretically exist in three different forms: a nonglycosylated one bearing an unmodified NX(S/T) sequence, a N-glycosylated one in which a glycan moiety is attached to Asn, and a de-N-glycosylated one resulting in the conversion of Asn to Asp. To analyze their fate as CTL epitopes, we synthesized peptides corresponding to the nonglycosylated (genetically encoded), N-glycosylated, and de-N-glycosylated forms of LCMV GP392-400 (named GP392-400, [GlcNAc-N396]-GP392-400, and [D396]-GP392-400 respectively), and of LCMV GP92-101 (named GP92-101, [GlcNAc-N95]-GP92-101, and [D95]-GP92-101 respectively). The N-acetyl-D-glucosamine moiety was chosen to mimic the glycosylated form because it is the first carbohydrate attached to Asn and common to all eukaryotic glycosylation processes. Previous studies have failed to reveal a response to LCMV GP392-400 following LCMV infection of mice (28), but in these studies the fact that GP392-400 bears a N-glycan in the mature viral protein was not taken into account. Our first aim was therefore to analyze the ability of the different unmodified (GP392-400) and post-translationally modified ([GlcNAc-N396]-GP392-400 and [D396]-GP392-400) forms of LCMV GP392-400 to generate a CTL response upon immunization. After several rounds of in vitro restimulation, CTL from peptide-immunized B6 mice were obtained against the GP392-400 form but not against the [GlcNAc-N396]-GP392-400 and [D396]-GP392-400 forms. This indicated that only GP392-400 was immunogenic, a result that contrasted with those obtained with LCMV GP92-101, where all three forms (GP92-101, [GlcNAc-N95]-GP92-101, and [D95]-GP92-101) were found to be immunogenic under the same experimental conditions (16). As shown in Fig. 1, anti-GP392-400 CTL specifically recognized the H-2b RMA (panel A) or MC57 (panel B) cells pulsed with GP392-400, in an effector:target (E:T) ratio-dependent manner. Unpulsed targets were not lysed (RMA) or were lysed weakly at the highest E:T ratios (MC57).


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  		Fig. 1.   GP392-400 is immunogenic in C57BL/6 mice. CTL were obtained from C57BL/6 mice immunized with 50-100 µg of peptides and restimulated as described under "Experimental Procedures." 51Cr-labeled RMA cells (A) or 51Cr-labeled MC57 cells (B) were pulsed with 10-6 M of synthetic GP392-400 (closed circles) or not (open circles) and were incubated with the anti-GP392-400 CTL line at the indicated E:T ratio for 4 h at 37 °C.

A CTL clone was then derived from the LCMV GP392-400-specific CTL line by limiting dilution. We tested the ability of these CTL to kill target cells coated with the immunizing peptide GP392-400 or its post-translationally modified forms [GlcNAc-N396]-GP392-400 or [D396]-GP392-400. Both the CTL line (Fig. 2A) and the CTL clone (Fig. 2B) recognized and lysed GP392-400-pulsed target cells with comparable efficacy (half-maximal lysis in the pM range). In contrast, 3-4-log higher concentrations of [GlcNAc-N396]-GP392-400 or of [D396]-GP392-400 (half-maximal lysis in the 10-9-10-8 M range) were required to obtain the same lysis by both the CTL line and the clone. In a comparative analysis shown in Fig. 2C, LCMV GP92-101-specific CTL generated against the GP92-101 peptide recognized the [D95]-GP92-101 form more efficiently than the GP92-101 form used for the immunization (half-maximal lysis at 10-12 and 10-10 M, respectively). Higher concentrations of [GlcNAc-N95]-GP92-101 (half-maximal lysis in the 10-8 M range, comparable with that of [GlcNAc-N396]-GP392-400 by anti-GP392-400 specific CTL) were necessary to sensitize target cells to CTL killing, confirming previous results (16).


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  		Fig. 2.   Ability of GP392-400-specific or GP92-101-specific CTL to recognize post-translationally modified analogs of GP392-400 or GP92-101. 51Cr-labeled RMA cells were pulsed with increasing concentrations (0-10-6 M) of synthetic peptides GP392-400 (black circles), [GlcNAc-N396]-GP392-400 (open circles), and [D396]-GP392-400 (gray circles) and were incubated with the anti-GP392-400 CTL line (A) or anti-GP392-400 CTL clone (B) at an E:T ratio of 10:1 for 4 h. A similar analysis was performed for GP92-101 (black squares), [GlcNAc-N95]-GP92-101 (open squares), and [D95]-GP92-101 (gray squares) testing recognition by a GP92-101-specific CTL line (C).

Post-translational Modifications (N-Glycosylation or De-N-glycosylation) Differentially Affect the Binding of GP392-400 and of GP92-101 to H-2Db-- Because one of the factors determining the immunogenicity of peptides is their ability to be presented by MHC class-I molecules (31, 32), we hypothesized that post-translational modifications could differentially affect the MHC class I presentation and could result in different immunogenic properties of the LCMV viral glycopeptides. Peptide binding to H-2Db was measured in a previously described MHC stabilization assay on RMA-S cells (25, 29). As shown in Fig. 3A, GP392-400 bound efficiently to H-2Db (half-maximal binding in the 0.1-0.2 µM range), in agreement with previous observations (25). In contrast binding of the two modified analogs to H-2Db was strongly reduced; [D396]-GP392-400 showed a 2-log decrease in H-2Db binding properties (half-maximal binding at 10 µM). Half-maximal binding was even not measurable for [GlcNAc-N396]-GP392-400 (only ~25% maximal binding was observed at the highest concentration tested (10-4 M)). Deficiency in MHC binding of these two peptides is therefore likely one of the reasons that may explain their lack of immunogenicity. A different situation was observed with the LCMV GP92-101 subdominant antigen for which the three forms bound efficiently to H-2Db (Fig. 3B). GP92-101 and [D95]-GP92-101 showed superimposed curves (half-maximal binding at <0.1 µM), whereas the [GlcNAc-N395]-GP92-101 form exhibited a 1-log higher binding (half-maximal binding at <0.01 µM). The differences in the efficiency with which they are recognized by CTL may thus likely reflect differences in TCR recognition rather than MHC binding (16).


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  		Fig. 3.   Binding of GP392-400 and GP92-101 and their glycosylated and de-glycosylated analogs to H-2Db. Binding to H-2Db was measured using a previously described stabilization assay. Increasing concentrations (0-10-4 M) of unmodified (black circles), N-glycosylated (open circles) and de-N-glycosylated (gray circles) forms of GP392-400 (A) or GP92-101 (B) were incubated for 4 h at 37 °C with RMA-S cells previously cultured for 40 h at 26 °C. After staining with the 28-14-8S anti- H-2Db mAb and a secondary antibody conjugated to fluorescein isothiocyanate, the cells were analyzed by flow cytometry. The results are representative of those obtained in three independent experiments.

LCMV-infected MC57 Cells Are Not Lysed by LCMV GP392-400- specific CTL-- It can be deduced from the experiments presented above that only the Asn396 form of GP392-400 is likely to be presented by H-2Db at the surface of infected cells, whereas all three forms of GP92-101 could be presented. The cytolytic activity of the anti-GP392-400 CTL was thus tested against LCMV-infected or uninfected MC57 target cells. Fig. 4 shows that neither the CTL line (panel A) nor the CTL clone (panel B) generated against GP392-400 were able to kill LCMV-infected cells, even at the highest E:T ratio tested. In comparison (panel C), efficient killing was observed when the CTL line generated against the unmodified form of the subdominant LCMV epitope GP92-101 was used, in accord with a previous study (16). Further, when HPLC-fractionated material eluted from the surface of LCMV-infected MC57 cells was used to pulse uninfected targets, no lysis by anti-GP392-400 CTL was observed (Fig. 5B). This observation contrasted that made with cells coated with mock-extracted synthetic GP392-400 peptide (Fig. 5A) and results obtained with anti-GP92-101 CTL directed against cells pulsed with the same pool of peptides extracted from LCMV-infected MC57 cells (16). Because a very low number of copies (100 or less) of an antigenic peptide present at the cell surface is sufficient to trigger CTL lysis, we can conclude from these results that GP392-400 is not naturally presented at the surface of LCMV-infected cells. This observation contrasts with that made with LCMV GP92-101 for which we found that both the genetically encoded GP92-101 and post-translationally modified [D95]-GP92-101 forms were co-presented to CTL at the surface of LCMV-infected cells, whereas the N-glycosylated form [GlcNAc-N95]-GP92-101 was absent (16).


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  		Fig. 4.   Recognition of LCMV-infected MC57 cells by anti-GP392-400 specific CTL. MC57 (H-2b) cells were previously infected (closed circles) or not (open circles) with LCMV (multiplicity of infection = 2) 48 h before the assay. 51Cr-labeled MC57 target cells were then incubated for 4 h with the anti-GP392-400 CTL line (A), anti-GP392-400 CTL clone (B), or anti-GP92-101 CTL (C) at the indicated E:T ratio. Specific lysis was measured as indicated under "Experimental Procedures."


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  		Fig. 5.   Recognition of acid-eluted peptides from LCMV-infected MC57 cells by anti-GP392-400-specific CTL. Each HPLC fraction corresponding to mock-extracted synthetic LCMV GP396-400 control peptide (solid circles, A) or to material eluted from the surface of LCMV-infected MC57 cells (2 × 108 eq) (open circles, B) was tested for its capacity to sensitize RMA cells to lysis by anti-GP392-400 CTL in a classical CTL assay as described under "Experimental Procedures." The arrows numbered 1, 2, and 3 indicate the retention times of synthetic peptides [GlcNAc-N396]-GP392-400, GP392-400, and [D396]-GP392-400, respectively. The results are expressed as percentages of specific lysis and are representative of two independent experiments.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In this study, by analyzing two H-2Db-restricted LCMV glycopeptides, GP392-400 and GP92-101 (25, 26), we showed that post-translational modifications of MHC class I-restricted viral peptides that bear a glycosylation motif NXS can have pleiotropic effects on their presentation by MHC and CTL recognition, depending on the peptide sequence (Table I). We previously reported that synthetic peptides corresponding to the unmodified (genetically encoded), N-glycosylated or de-N-glycosylated analogs of GP92-101 share the ability to bind strongly to H-2Db, are both immunogenic, and are naturally processed and presented at the surface of infected cells (16). We now show that post-translational modifications of so called "high affinity peptides" do not always have a positive effect on the diversity of antigen presentation. The results presented in this paper show that N-glycosylation or de-N-glycosylation of GP392-400 led to peptides with reduced MHC binding properties at physiological concentrations, whereas unmodified GP392-400 efficiently bound to this molecule. This may be one of the reasons why only the unmodified form of GP392-400 is immunogenic in C56BL/6 mice and could generate epitope-specific CTL that efficiently lysed peptide-pulsed target cells. However GP392-400-specific CTL did not recognize LCMV-infected cells, showing that GP392-400 is not presented by H-2Db in a natural situation. The lack of presentation of the [GlcNAc-N396]-GP392-400 and [D396]-GP392-400 forms can be easily explained by our observation that those peptides are poor H-2Db binders and show no immunogenicity. Indeed above a certain threshold of binding to the MHC, the immunogenicity of peptides rapidly drops, preventing these peptides from acting as T cell epitopes (33). That LCMV-infected cells were not recognized by anti-GP392-400 CTL tells us that not only [GlcNAc-N396]-GP392-400 and [D396]-GP392-400 but also GP392-400, despite its high H-2Db binding properties, were absent from the infected cell surface.

                              
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Table I
Summary of the cellular, biochemical, and immunological properties of the different forms of the viral glycopeptides LCMV GP392-400 and GP92-101

Why is the genetically encoded form of GP92-101 but not that of GP392-400 naturally presented at the surface of infected cells, whereas both GP92-101 and GP392-400 are known to be N-glycosylated in their respective proteins GP1 and GP2 (27)? One explanation is that some of the glycosylation sites of LCMV GP1 but none in GP2 may be statistically left unglycosylated within the viral glycoprotein. Conversely it is conceivable that some GP1 but not GP2 is aberrantly translated on free ribosomes in the cytosol (34). Whatever the explanation, GP2 protein containing Asn at position 396 would require further processing mediated by the proteasome before being presented by MHC class I molecules. Interestingly we observed that purified proteasomes were unable to cleave the GP392-400 precursor at the correct C terminus of the potential CTL epitope, therefore suggesting that even though position 396 was left unmodified, no production of GP392-400 would occur.2 The inability of the proteasome to generate an antigenic peptide or a precursor extended by a few amino acids at its N terminus has been shown to represent one of the main blocks in antigen presentation (5-7).

What may explain the differences between GP92-101 and GP392-400 with regard to the effect of post-translational modifications? In terms of MHC presentation, the loss of MHC binding capacity of the [GlcNAc-N396]-GP392-400 and [D396]-GP392-400 forms of GP392-400 is likely to be due to the role played by Asn396 as an MHC anchor (Asn at position 5 of the sequence for H-2Db). Indeed we and others have previously shown that one important property of MHC anchor residues is their limited or null tolerance to substitution (29, 35, 36). However, in some instances glycosylation of residues at anchor position has been shown to preserve the ability of the modified peptide to bind to MHC molecules and to be recognized by CTL (9, 11). In the case of GP92-101, this peptide contains two adjacent N residues. It is likely that Asn96 and not Asn95 serves as the H-2Db anchor because: (i) lengthening GP92-101 at the N terminus by one amino acid (GP91-101) that shifted Asn95 to position 5 of the sequence resulted in a marked loss of H-2Db binding (25) as would be expected if Asn96 is the anchor residue (an increase in H-2Db binding would have been expected if N95 played this role) and (ii) the high H-2Db binding properties of GP92-101 were left unaffected by either N-glycosylation or de-N-glycosylation that in turn dramatically altered TCR recognition, consistent with previous data showing that position 4 of H-2Db-restricted peptides tolerates a wide variety of substitutions and is directed toward the TCR. This is well supported by three-dimensional structure and molecular models of GP392-H-2Db and GP92-H-2Db complexes, which clearly place position 4 and position 5 as TCR contact and MHC anchor residues respectively (Fig. 6). Also the data presented in Figs. 2 and 3 show that the CTL recognition properties of GP392-400, [GlcNAc-N396]-GP392-400, and [D396]-GP392-400 forms of GP392-400 were directly correlated to their MHC binding properties, whereas the CTL recognition of GP92-101, [GlcNAc-N95]-GP92-101, and [D95]-GP92-101 were not. Therefore, the beneficial or detrimental effects of post-translational modification of peptides on T cell activation will greatly depend on the nature and location of the acceptor site within the peptide sequence (Refs. 9, 11, and 16 and this report). If beneficial, these modifications may be seen as an advantage to the host in its fight against viral infection by creating neo-epitopes (16). Conversely, if detrimental, they may be considered as a means for a virus to achieve CTL escape by destroying potential immunogenic sequences (37, 38).


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  		Fig. 6.   Molecular models of LCMV GP92-101 and GP392-400 complexed with H-2Db. The three-dimensional models for LCMV GP92-101 (CSANNSHHYI) (A) and LCMV GP392-400 (WLVTNGSYL) (B) interacting with H-2Db have been produced using the online modeling facility SwissModel (GlaxoWellcome) at the following address: www.expasy.ch/spdbv/. The three residues Asn, Xaa, and Ser of the NXS glycosylation motif are shown in red (Asn95 or Asn396 as acceptor residue of N-glycosylation and de-N-glycosylation), yellow (Asn96 or Gly397), and blue (Ser97 or Ser298), respectively. The arrows indicate the H-2Db anchor positions. Post-translational modifications of Asn95 do not affect MHC presentation of GP92-101 but influence T cell recognition (this study and Ref. 16), whereas post-translational modifications of Asn396 are detrimental to H-2Db binding of GP392-400.

It is interest to note that whereas artificially N-glycosylated model peptides can efficiently bind classical MHC class I molecules and be recognized by CTL (8-12), no such naturally processed N-glycopeptides have yet been identified. However, natural peptides bearing a N-glycosylation motif appeared, as presented by classical MHC class I molecules, at the surface of target cells in their nonglycosylated or de-N-glycosylated forms (16-18). In contrast several N-glycopeptides were found to be naturally presented by MHC class II or nonclassical MHC class I (39-49). In addition, experimental evidence exists that O-glycosylated peptides (50) or peptides bearing other post-translational modifications such as cysteinylation (51-53) or phosphorylation (19) can be naturally presented by classical MHC class I. It is possible that N-glycopeptides could not be naturally presented as a result of constraints imposed by the processing pathway (54, 55). Altogether key parameters that contribute to the creation or conversely to the disruption of potent antiviral epitopes such as the ones presented here must be taken into account in approaches to predicting viral or tumor antigens of therapeutic interest.

    ACKNOWLEDGEMENTS

We thank S. Claverol and B. Monsarrat for sharing unpublished observations, P. Borrow for helpful discussions and critical reading of the manuscript, and Y. Barascud and C. Grégoire for help in MHC binding assays.

    FOOTNOTES

* This work was supported in part by a grant from the Center National de la Recherche Scientifique and by l'Association pour la Recherche sur le Cancer Contract 5485.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Recipient of a post-doctoral fellowship from the Association pour la Recherche sur le Cancer.

|| To whom correspondence should be addressed: Laboratoire d'ImmunoPharmacologie Structurale, Institut de Pharmacologie et de Biologie Structurale, 205 route de Narbonne, 31400 Toulouse, France. Tel.: 33-561-175-530; Fax: 33-561-175-532; E-mail: gairin@ipbs.fr.

2 S. Claverol, B. Monsarrat, and J. E. Gairin, unpublished observations.

Published, JBC Papers in Press, DOI 10.1074/jbc.M105974200

    ABBREVIATIONS

The abbreviations used are: MHC, major histocompatibility complex; LCMV, lymphocytic choriomeningitis virus; CTL, cytotoxic T lymphocyte(s); Fmoc, N-(9-fluorenyl)methoxycarbonyl; HPLC, high pressure liquid chromatography; BSA, bovine serum albumin; PBS, phosphate-buffered saline; E:T, effector:target.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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