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Genetically encoded and post-translationnally modified
forms of a major histocompatibility complex class-I
restricted antigen bearing a glycosylation motif are
independently processed and co-presented to cytotoxic
T lymphocytes.
Denis Hudrisier, Joelle Riond, Honore Mazarguil, Michael B. Oldstone, Jean
Edouard Gairin
To cite this version:
Denis Hudrisier, Joelle Riond, Honore Mazarguil, Michael B. Oldstone, Jean Edouard Gairin.
Genet-ically encoded and post-translationnally modified forms of a major histocompatibility complex class-I
restricted antigen bearing a glycosylation motif are independently processed and co-presented to
cy-totoxic T lymphocytes.. Journal of Biological Chemistry, American Society for Biochemistry and
Molecular Biology, 1999, 274, pp.36274-36280. �hal-00092655�
Genetically Encoded and Post-translationally Modified Forms of a
Major Histocompatibility Complex Class I-restricted Antigen
Bearing a Glycosylation Motif Are Independently Processed
and Co-presented to Cytotoxic T Lymphocytes*
(Received for publication, July 12, 1999, and in revised form, September 13, 1999) Denis Hudrisier‡§, Joe¨lle Riond‡, Honore´ Mazarguil‡, Michael B. A. Oldstone¶,
and Jean Edouard Gairin‡
From the ‡Institut de Pharmacologie et de Biologie Structurale, UPR 9062 CNRS, 205 route de Narbonne, 31400 Toulouse, France and the¶Department of Neuropharmacology, Scripps Research Institute, La Jolla, California 92037
The mechanisms by which antigenic peptides bearing a glycosylation site may be processed from viral glyco-proteins, post-translationally modified, and presented by major histocompatibility complex class I molecules remain poorly understood. With the aim of exploring these processes, we have dissected the structural and functional properties of the MHC-restricted peptide GP92–101 (CSANNSHHYI) generated from the lympho-cytic choriomeningitis virus (LCMV) GP1 glycoprotein. LCMV GP92–101 bears a glycosylation motif -NXS- that is naturally N-glycosylated in the mature viral glycopro-tein, displays high affinity for H-2Dbmolecules, and elic-its a CD81cytotoxic T lymphocyte response. By analyz-ing the functional properties of natural and synthetic peptides and by identifying the viral sequence(s) from the pool of naturally occurring peptides, we demon-strated that multiple forms of LCMV GP92–101 were generated from the viral glycoprotein and co-presented at the surface of LCMV-infected cells. They corre-sponded to non-glycosylated and post-translationally modified sequences (conversion of Asn-95 to Asp or al-teration of Cys-92). The glycosylated form, despite its potential immunogenicity, was not detected. These data illustrate that distinct, non-mutually exclusive antigen presentation pathways may occur simultaneously within a cell to generate structurally and functionally different peptides from a single genetically encoded se-quence, thus contributing to increasing the diversity of the T cell repertoire.
Cytotoxic T lymphocytes (CTL)1 recognize, via their T cell
receptor (TCR), molecular complexes formed between major
histocompatibility complex (MHC) class I molecules and short antigenic peptides generated from a multistep intracellular processing of viral proteins involving components present in the cytoplasm and the endoplasmic reticulum (ER). Processing of antigenic peptides from cytosolic or nuclear proteins has been extensively studied (for review, see Ref. 1). It begins in the cytosol with the breakdown of ubiquitinated proteins by the proteasome and possibly by nonproteasomal cytosolic proteases (2– 4). Peptides generated by this degradation step are then translocated via peptide-specific transporters (TAP) into the ER, where association with newly synthesized MHC class I (5) and, eventually, further trimming of peptides (6, 7) occur.
In contrast, the mechanisms by which MHC-restricted anti-genic peptides are generated from transmembrane or secreted glycoproteins, which are co-translationally translocated into the ER during biosynthesis, are less understood; however, they frequently represent a source of antigenic peptides (8). The observation that, in most cases, their processing requires both proteasome activity and TAP-dependent translocation into the ER suggests that at least a certain amount of the glycoprotein may have access to the cytosol (9 –11). Both mistranslation in the cytoplasm (12) and retrograde transport from the ER to the cytosol have been proposed to explain the presence of glycopro-teins in the cytosol (11, 13).
It is thus of particular interest to study the processing from viral glycoproteins of peptides bearing a glycosylation motif (N-X-S/T, where X is any amino acid except P). In theory, such peptides can exist in diverse forms depending on their post-translational state: non-glycosylated, N-glycosylated, or de-N-glycosylated. These forms are characterized, respectively, by the presence at the level of the N-X-S/T motif of an unmodified N, a glycan-branched N or a D (as the result of a peptide:N-glycanase activity that provokes deamidation of the side chain (14)). Further, the enzymatic activities required for N-glycosy-lation (N-glycosyltransferases) and de-N-glycosyN-glycosy-lation (pep-tide:N-glycanase) are thought to be located in the ER/Golgi (15) and cytosol/nucleus (14), respectively.
Infection of H-2b mice with lymphocytic choriomeningitis
virus (LCMV) generates a vigorous CD81 CTL response against three well defined H-2Db-restricted epitopes located in
the glyco- (GP1 and GP2) and nucleo- (NP) proteins (16 –19). By screening these viral proteins for potential new epitopes, the LCMV GP92–101 (CSANNSHHYI) peptide was identified first as a strong H-2Db binder (20, 21), and later as a
non-immu-nodominant LCMV epitope (22). Interestingly, the central core motif GP95–97 of this antigen represents one of the glycosyla-tion motifs, -NXS/T-, known to be N-glycosylated in the mature LCMV GP1 glycoprotein (23). These observations at both the
* This work was supported in part by Association pour la Recherche sur le Cancer Contract 9261, the Comite´ Re´gional de Haute-Garonne de la Ligue Nationale contre le Cancer, and Conseil Re´gional de Midi-Pyre´ne´es Contract 9609711. 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.
§ Recipient of a postdoctoral fellowship from the Association pour la Recherche sur le Cancer. To whom correspondence should be addressed. Tel.: 33-0-561-175-531; Fax: 33-0-561-175-994; E-mail: hudrisie@ipbs. fr.
1The abbreviations used are: CTL, cytotoxic T lymphocyte; MHC, major histocompatibility complex; TCR, T cell receptor; HPLC, high performance liquid chromatography; LCMV, lymphocytic choriomenin-gitis virus; ER, endoplasmic reticulum: GlcNac, N-acetyl-D -glucosa-mine; DTT, dithiothreitol; FITC, fluorescein isothiocyanate; EndoH, endoglycosidase H; GP, glycoprotein; NP, nucleoprotein; PBS, phos-phate-buffered saline; E:T, effector:target; Fmoc, N-(9-fluorenyl)me-thoxycarbonyl; TAP, peptide-specific transporter.
© 1999 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.
This paper is available on line at http://www.jbc.org
36274
This is an Open Access article under the CC BY license.structural and functional levels make this peptide a good model for studying the effect of post-translational modification on antigen processing and presentation, and the consequences it may have for the antiviral CTL response. This study aimed: (i) to analyze the effect of post-translational modification (N-gly-cosylation and de-N-gly(N-gly-cosylation) on the MHC binding and CTL activation properties of LCMV GP92–101 and (ii) to iden-tify the viral sequence(s) naturally processed and presented to CTLs at the surface of LCMV-infected cells. Binding studies performed with synthetic peptides corresponding to the three possible forms of GP92–101 indicated that neither N-glycosy-lation nor de-N-glycosyN-glycosy-lation affected the H-2Db-binding
affin-ity of GP92–101. All three forms of the peptide were immuno-genic and led to the generation of GP92–101-specific CTLs. Analysis of peptides eluted from the surface of LCMV-infected cells by a cytolytic assay coupled to HPLC led to the identifi-cation of both non-glycosylated (N95) and de-N-glycosylated sequences (D95) naturally presented at the surface of infected cells. The N-glycosylated (GlcNac-N95) form remained unde-tectable. Additionally, immunoreactive species of higher reten-tion time derived from the N95 peptide were also observed, likely corresponding to oxidized forms of C92. The physiological relevance of this remains to be determined. Overall, the data presented here demonstrate for the first time that both genet-ically encoded and post-translationally modified forms of a viral glycopeptide may co-exist at the surface of virus-infected cells as a result of distinct, non-mutually exclusive processing pathways that can operate simultaneously.
MATERIALS AND METHODS
CTL, Cell Lines, and Virus—CTL were obtained from C57BL/6 mice
immunized subcutaneously at the base of the tail with 50 –100mg of the indicated peptides mixed with 5mg of P30 T-helper epitope from teta-nus toxoid (24) in incomplete Freund’s adjuvant. After 1 week, draining lymph nodes were removed and CTL were restimulated weekly with irradiated (2,500 rads) C57BL/6 splenocytes and irradiated (10,000 rads) peptide-pulsed (1mM) RMA cells in the presence of 30 UI/ml IL2 (EL4 supernatant). Murine H-2bmutant RMA-S cells and human T2 cells transfected with H-2Db(T2-Db) were used in binding experiments. The murine H-2bcell lines RMA and MC57 were used in in vitro cytotoxicity assays. Cells were grown in Dulbecco’s modified Eagle’s medium (RMA, RMA-S, MC57) or Iscove’s modified Dulbecco’s medium (T2-Db) containing 5% bovine serum,
L-glutamine (2 mM) and antibiotics (10 units/ml penicillin and 10 mg/ml streptomycin). Geneticin (400 mg/ml) was added to Iscove’s modified Dulbecco’s medium to maintain selection of T2-Dbcells. Lymphocytic choriomeningitis virus Armstrong strain (LCMV Arm) was used to infect mice or cells.
Peptide Synthesis—Peptides were synthesized on an automated
pep-tide synthesizer (Applied Biosystems 430A) by the solid-phase method using Fmoc chemistry. The glycopeptide analogue was obtained by using Fmoc-Asn(Ac3AcNH-bGlc)-OH, a commercially available deriva-tive of Asn bearing an N-acetyl-D-glucosamine moiety (Novabiochem).
After standard protocols for solid phase synthesis, cleavage, and depro-tection, the glycopeptide was de-O-acetylated with 0.1Msodium
hydrox-ide. Peptides were purified by HPLC on a RP300-C8 reversed-phase column (Brownlee Lab) and their identity confirmed by electrospray ionization mass spectrometry.
Binding Studies—Binding studies were performed as described
pre-viously (20). Briefly, T2-Dbcells (13 105cells/well) were incubated in U-bottomed 96-well plates for 75 min at 37 °C with 100 nMH-2Db
-restricted fluorescent probe FITC-KAIENAEAL (25) and increasing concentrations (10210to 1024M) of unlabeled competitors. Cells were then washed twice with bovine serum albumin-PBS and analyzed by flow cytometry. Total and nonspecific binding were measured in the absence or presence of 1 mMunlabeled LCMV NP396 – 404. Specific binding to H-2Dbwas defined as the difference between the total and nonspecific binding. Percentage (%) of binding inhibition was calculated as 1003 {1 2 (fluorescence intensity (FI) in the presence of competi-tor2 FI, nonspecific binding/FI, specific binding)}. IC50represents the peptide concentration inhibiting 50% of the specific binding of the fluorescent probe.
Extraction of Viral Peptides from Infected Cells—MC57 (H-2b) and control Balb/c (H-2d) cell lines (1–23 109cells) were infected or not with
LCMV Arm for 48 h. Cells were washed three times in PBS, then resuspended in 0.1Mcitrate/phosphate buffer at pH 3.0 for 2 min. The eluted material was desalted on a Sep-Pak column (Waters) according to the manufacturer’s instructions, then transferred onto a Centricon 10 (Amicon) and centrifuged at 3500 rpm for 90 min at 4 °C. Material less than 10 kDa was vacuum-concentrated and then resuspended in 20 ml of 0.08% trifluoroacetic acid. Peptide separation was carried out on a reversed-phase C18 column (7mm, 2.1 3 100 mm, Aquapore, Brownlee) using the Waters 600S controller system. Samples (10ml) were injected and separated using either system I (5– 60% B in 60 min) or system II (6 –15% B in 30 min, then 15–57% B in 30 min). Solution A was 0.08% trifluoroacetic acid in H2O, and solution B was 0.08% trifluoroacetic acid in CH3CN (flow rate: 400ml/min). Fractions (200 ml) were collected in U-bottomed 96-well plates, lyophilized, reconstituted in PBS, and stored at280 °C before analysis. In some cases, the eluted pool was treated with DTT (1 mMDTT in 0.1Mammonium acetate, pH 8.5, under N2overnight at room temperature) or Endo H (50 milliunits/ml, pH 5.5, 24 h at 37 °C) before separation.
Cytotoxicity Assays—RMA cells were incubated for 1 h at 37 °C in
medium containing 10-fold dilutions of peptides. MC57 cells were in-fected at a multiplicity of infection of 2 with LCMV Arm 48 h prior the assay. Peptide-pulsed and control RMA cells, and LCMV-infected and uninfected MC57 cells were 51Cr labeled and used as targets (5 3 103/well) in chromium release assays. Effector CTL were added at 1.53 104/well or at the indicated E/T ratio. The51Cr content of supernatants was determined after 4 h of incubation at 37 °C. The specific lysis was calculated as 100 3 [(experimental 2 spontaneous release)/(total 2 spontaneous release)].
RESULTS
N-Glycosylation and De-N-glycosylation Do Not Alter H-2Db
Binding Affinity of LCMV GP92–101—Three synthetic pep-tides corresponding to the non-glycosylated (N95), N-glycosy-lated ((GlcNac)-N95) and de-N-glycosyN-glycosy-lated (D95) forms of GP92–101 were synthesized (Table I). The N-acetyl-D -glucosa-mine moiety was chosen to mimic the glycosylated form be-cause: (i) it is the first carbohydrate attached to N and is common to all eukaryotic glycosylation processes and (ii) it corresponds to the minimal sugar moiety left after Endo H digestion of glycosylated side chains, therefore allowing the detection of natural species. Peptide binding affinities for H-2Dbwere measured in a MHC binding competition assay on
T2-Dbcells (25). As shown in Fig. 1, N95 bound to H-2Dbwith
a high affinity (IC50in the 10 nMrange), in agreement with
previous observations (20). The two modified analogues, (Glc-Nac)-N95 and D95, bound efficiently to T2-Dbcells with
dose-response curves superimposable on that of the unmodified N95, thus indicating that all three forms of GP92–101 can poten-tially be presented by H-2Dbat the surface of LCMV-infected
cells expressing H-2Db.
N95, (GlcNac)-N95, and D95 Forms of LCMV GP92–101 Generate a CTL Response in H-2b Mice—C57BL/6 mice were
immunized subcutaneously at the base of the tail with N95, (GlcNac)-N95, and D95 in the presence of the P30 helper epitope and incomplete Freund’s adjuvant. After 2 weeks of in vitro restimulation, CTL were obtained against each of the three peptides, indicating that they are all immunogenic. As shown in Fig. 2 (panel A), CTL generated against N95 killed target cells coated with the immunizing peptide with a half-maximal lysis at concentrations in the 1029to 10210Mrange. These CTL also recognized (GlcNac)-N95 and D95, but at very different concentrations. (GlcNac)-N95 recognition required a concentration about 2 logs higher, and the maximal lysis pla-teau reached was lower than that for N95, whereas D95 was ;100-fold more potent than N95. CTL generated against (Gl-cNac)-N95 (panel B) and D95 (panel C) showed a 10-fold higher efficacy (half-maximal lysis in the range of 10211 to 10210M range) and were fully specific for the immunizing peptide since no (or very weak) cross-reactivity with the other forms of GP92–101 was observed. The response generated against each form of the LCMV GP92–101 was peptide-specific since none of
the CTLs recognized the unrelated GP33– 41 sequence of the LCMV GP1 immunodominant epitope, even at the highest con-centration tested (1026M). Altogether, these results show that the three different forms of GP92–101 may represent distinct antigens. Obtention of these anti-GP92–101 CTLs then allowed us to search for the presence of GP92–101 sequence(s) natu-rally processed and presented at the surface of LCMV-infected cells.
LCMV-infected MC57 Cells Are Lysed by CTL Specific for Unmodified (N95) and Post-translationally Modified (D95) An-alogs of LCMV GP92–101—The cytolytic activity of the anti-GP92–101 was tested against LCMV-infected or uninfected MC57 target cells. Fig. 3 shows that CTL generated against N95 (panel A) and D95 (panel C) but not (GlcNac)-N95 (panel B) were able to specifically lyse LCMV-infected cells,
demon-strating that GP92–101 is naturally presented at the surface of LCMV-infected cells. This result is in agreement with the find-ing that anti-GP92–101-specific CTL are present in secondary antiviral CTL obtained after acute infection of C57BL/6 mice with LCMV (22, 26). Given the pattern of reactivity of N95- and D95-specific CTL (see Fig. 1A), one can deduce that at least the post-translationally modified form (D95) of LCMV GP92–101 is naturally presented at the surface of LCMV-infected cells.
Both Unmodified (N95) and Post-translationally Modified (D95) Forms of LCMV GP92–101 Are Naturally Presented at the Surface of LCMV-infected Cells—Since LCMV-infected cells were recognized and lysed by anti-LCMV GP92–101 CTLs, peptides were extracted from the surface of LCMV-infected (or as a control, uninfected MC57 cells), separated by HPLC (Fig. 4, panel A) and analyzed in cytotoxicity assays (Fig. 4, panel B), in order to characterize further which form(s) of LCMV GP92– 101 was(were) endogenously presented.
In a first step, CTL generated against N95 were used since they recognized all forms of GP92–101. As shown in Fig. 4 (panel B), these CTL killed H-2btarget cells coated with
ma-terial eluted from LCMV-infected but not uninfected MC57 cells. Two peaks of activity corresponding to fractions 31–34 (retention time: 15–16.5 min) and to fractions 36 –37 (retention time: 17.5–18.0 min) were obtained, indicating that different forms of GP92–101 were processed in LCMV-infected cells and presented at their surface. N95-specific CTL did not recognize peptides eluted from LCMV-infected or uninfected Balb/c (H-2d) cells in control experiments (data not shown).
In a second step, fractions 20 –50 from an independent ex-periment were analyzed with either N95- or D95-specific CTL, both of which recognized LCMV-infected cells. As shown in Fig. 5 (panels A and B), a different profile of activity was observed with the two CTL lines. The D95-specific CTL activity profile (panel B) was very clear and restricted to fraction 32, which, as indicated by the arrow, corresponds to the retention time of the synthetic D95 peptide. This result confirms unambiguously that the D95 form of GP92–101 is present at the surface of LCMV-infected cells. The activity profile obtained with N95-specific CTL (panel A) was more complex and showed that other forms of GP92–101 were present that were selectively recognized by N95-specific CTL but not D95-specific CTL.
Since the poor separation of the N95 and D95 forms obtained with the HPLC system I (see Table I) did not allow unambig-uous identification of the peptide(s) present in the main peak recognized by anti-N95 CTL (fractions 31–34, Fig. 5A), we set up an optimized gradient (system II) allowing a better separa-tion of N95 and D95 (see Table I). As shown in Fig. 6,
endog-TABLE I
Identification and physicochemical properties of the different synthetic forms of the non-immunodominant epitope GP92–101 of LCMV used in this study
Peptides were synthesized by solid-phase method as described under “Materials and Methods.”
Peptide Form Sequence Cellular statusa HPLC retention time b
Molecular weightc System I System II
min
N95 Non-glycosylated CSANNSHHYI Genetically encoded 16.2 15.0 1145.9
CSANNSHHYI (N95)2 Non-glycosylated dimer ?
CSANNSHHYI
Unknown 21.5 Not done 2289.4
GlcNac Glycosylated in the mature viral protein
(GlcNac)-N95 N-Glycosylated ?
CSANNSHHYI
13.5 Not done 1474.9
D95 De-N-glycosylated CSADNSHHYI Post-translationally modified 16.5 16.0 1146.7
aThe putative status of the corresponding endogenous sequence of the epitope in the LCMV infected cells is indicated. The two naturally
occurring forms identified in this study are underlined.
bThe HPLC systems I and II are as described under “Materials and Methods.”
cPeptides were identified by on-line HPLC ESI mass spectrometry. The values indicated correspond to the measured [M1 H]1species.
FIG. 1. H-2Dbbinding affinity of GP92–101 and its glycosylated and de-glycosylated analogs. Binding affinity to H-2Db
was meas-ured in a competition assay as described previously (20). Increasing concentrations (10210to 1025 M) of N95 (solid circles), (GlcNac)-N95 (dashed circles), D95 (open circles) and, as negative control, the H-2Ld -restricted peptide LCMV NP118 –126 (solid squares) were incubated with T2-Dbcells in the presence of 100 n
Mfluorescent H-2Db-specific
probe FITC-KAIENAEAL (25) for 75 min at 37 °C. After washing, cells were analyzed by flow cytometry. Results are expressed as percentage of inhibition of FITC-KAIENAEAL binding to T2-Db
and are represent-ative of three independent experiments.
enous viral material was then clearly detected in fractions 30 –31 by anti-N95 CTL but not by anti-D95 CTL. These frac-tions corresponded to the retention time of synthetic N95 iden-tified by UV detection and mass spectrometry analysis in the same experimental conditions (Table I). Fractions 32–34
rec-ognized by the two CTL lines contained the D95 form, as expected from results presented above and in Table I. Thus both the N95 and D95 forms of GP92–101 are naturally found at the surface of LCMV-infected H-2bcells.
The N-Glycosylated Form of GP92–101 Is Not Detected at the
FIG. 2. Characterization of N95-, (GlcNac)-N95-, and D95-specific CTL lines generated from immunized C57BL/6 mice. CTL were obtained from C57BL/6 mice immunized with 50 –100 mg of peptides and restimulated as de-scribed under “Materials and Methods.” 51Cr-labeled RMA cells were pulsed with increasing concentrations (10213to 1026
M) of synthetic peptides N95 (solid
cir-cles), (GlcNac)-N95 (dashed circir-cles), D95
(open circles) and, as a control, GP33– 41 (KAVYNFATC) (solid squares) and were incubated with CTL specific for anti-N95 (panel A), anti-(GlcNac)-N95 (panel B), and anti-D95 (panel C) at an E:T ratio of 10:1 for 4 h.
FIG. 3. Recognition of LCMV-in-fected MC57 cells by N95-, anti-(GlcNac)-N95-, and anti-D95-specific CTL. MC57 (H-2b) cells were uninfected (open circles) or infected (solid circles) with LCMV (multiplicity of infection5 2) 48 h before the assay.51Cr-labeled MC57 target cells were then incubated for 4 h with CTL specific for anti-N95 (panel A), (GlcNac)-N95 (panel B), and anti-D95 (panel C) at various E:T ratio. Spe-cific lysis was measured as indicated un-der “Materials and Methods.” Results are representative of at least two independ-ent experimindepend-ents.
FIG. 4. Recognition of acid-eluted peptides from LCMV-infected cells by anti-N95 CTL. A, material acid-eluted from the surface of MC57 cells was fractionated on RP-HPLC using gradient system I. B, Each HPLC fraction corre-sponding to material eluted from the cell surface of LCMV-infected (solid circles) or uninfected (open circles) MC57 cells (23 108cell eq) was tested for its capacity to sensitize RMA cells to lysis by anti-N95 CTL in a classical CTL assay as described under “Materials and Methods.” Results are expressed as percentage (%) of specific lysis, and are representative of two inde-pendent experiments.
Surface of LCMV-infected Cells—Fractions 36 –37 recognized by N95-specific CTL do not co-migrate with any of the synthetic peptides corresponding to post-translationally modified ana-logs of GP92–101. We reasoned that this peak could correspond to a N-glycosylated form of GP92–101 different from the syn-thetic (GlcNac)-N95 modified peptide and thus not recognized by (GlcNac)-N95-specific CTL. To investigate this possibility, we treated the pool of eluted peptides with Endo H, which generates the (GlcNac)-N form from complex glycans attached to N, before performing the assay. Despite this treatment, none of the fractions was recognized by (GlcNac)-N95-specific CTL (shown in Fig. 7), indicating that no N-glycosylated form of
GP92–101 was detectable among the eluted peptides. The syn-thetic control (GlcNac)-N95 peptide was recognized by specific CTL in fractions corresponding to the expected retention time. Fractions 36 –37 Correspond to an Oxidized C92 Derivative of the N95 Form of LCMV GP92–101—The presence of a cysteinyl residue (C92) in the GP92–101 sequence may lead to oxidized species as a result of either antigen processing or storage of peptide eluates (27). As shown in Fig. 8, DTT treatment of peptide eluate abrogated the CTL activity against fractions 36 – 40. To support this observation, we found that the dimer-ized N95 form of the GP92–101 synthetic peptide which eluted at an expected higher retention time (Table I) was still effi-ciently recognized by N95-specific CTL (data not shown). Thus, the DTT-reducible, N95-specific immunoreactivity found in fractions 36 – 40 reflects the presence within the peptide eluate of chemically modified N95 forms of LCMV GP92–101 which likely correspond to oxidized C92 derivatives. Why oxidation of C92 only affected N95 and not D95 is unknown.
Altogether our results indicate that GP92–101 is present at the surface of infected cells as at least two different endog-enously processed forms, a non-glycosylated one and a de-N-glycosylated one. A third one corresponding to a chemical mod-ification of C92, whose natural occurrence remains to be established, was also detected.
DISCUSSION
In this study, we demonstrate, using the non-immunodomi-nant H-2Db-restricted LCMV epitope GP92–101, that MHC
class I-restricted antigens that bear a glycosylation motif -NXS- may be processed from a viral glycoprotein and pre-sented at the surface of infected cells as at least two distinct sequences, genetically encoded (non-glycosylated) and post-translationally modified (de-N-glycosylated).
To our knowledge, such a dual processing and presentation pathway has never been observed before. Very few (three) examples of naturally processed sequences bearing a glycosy-lation motif have been reported so far, consisting in each case of only one exclusive form, either non-glycosylated (28) or
de-FIG. 5. Comparative recognition by anti-N95 and anti-D95 CTL of pep-tides extracted from LCMV-infected cells. HPLC fractions (gradient system I) 20 –50 corresponding to material eluted from LCMV-infected (solid circles) and uninfected (open circles) MC57 (108cell eq) were tested as described in the legend of Fig. 4 using N95 (panel A) or anti-D95 (panel B) CTL at an E:T ratio of 10:1.
Arrows indicate the retention time of
syn-thetic peptides N95 (panel A) and D95 (panel B).
FIG. 6. Fine resolution of LCMV peptides recognized by anti-N95 and anti-D95 CTL. HPLC fractions 20 –50 corresponding to ma-terial eluted from LCMV-infected MC57 cells (108cell eq) were tested as described in the legend of Fig. 4 except that gradient system II was used. Fractions (data is only shown for fractions 27–36) were tested for their ability to sensitize RMA cells to lysis by anti-N95 (solid circles) or anti-D95 (open circles) CTL at an E:T ratio of 10:1.
N-glycosylated (10, 29). How may the two presentation path-ways of LCMV GP92–101 be achieved? For the de-N-glycosy-lated form, processing might involve translation in the ER, export of full-length glycoprotein to the cytosol and transport of processed peptides by TAP for MHC binding, as shown for an HLA-A2 tyrosinase epitope (11). Proteasomal degradation of glycoproteins requires their retro-transport from the endoplas-mic reticulum (where glycosylation occurs) to the cytosol (where both deglycosylation and degradation are achieved) (30, 31). With regard to the non-glycosylated form, it is difficult to understand how a non-glycosylated peptide may be processed from the LCMV GP1 glycoprotein itself since all the five glyco-sylation motifs -NXS/T- of LCMV GP1 are glycosylated in the mature viral glycoprotein (23). Further, the
peptide:N-gly-canases known so far, due to their b-aspartyl-glycosylamine hydrolase activity, always convert the glycosylated Asn to Asp and never restore the Asn initially present. One likely expla-nation is that the non-glycosylated form of LCMV GP92–101 does not originate from GP1 but from LCMV GP-C. The LCMV glycoprotein GP1 is generated, together with GP2, from a post-translational cleavage of a common precursor GP-C. Unglyco-sylated GP-C is not cleaved (23); one may therefore hypothesize that the non-glycosylated form of GP92–101 originates from unglycosylated, uncleaved GP-C and not from GP1. The non-glycosylated and the de-N-non-glycosylated forms of GP92–101 would then be generated separately from two different pro-teins. Two other possibilities can also be proposed (28): (i) some of the glycosylation sites of LCMV GP1 may be randomly left unglycosylated within the viral glycoprotein, or (ii) parts of the glycoprotein may be aberrantly translated on free ribosomes in the cytosol and thus never access the ER. Determination of the relative proportion of the different forms of GP92–101 pre-sented at the surface of infected cells would require highly sensitive detection methods not available in our laboratory. It is expected that the proportion of the different forms we ob-served would be determined by both (i) the efficacy of the processing pathways involved and (ii) the initial amount of substrate for these different pathways.
While the natural occurrence of glycosylated peptides pre-sented by MHC class II molecules to abTCR has been well documented (32, 33), the same remains to be demonstrated for classical MHC class I molecules. The presence of the de-N-glycosylated form of GP92–101 clearly indicates that a certain proportion of the peptide has previously been N-glycosylated, yet we were unable to detect any N-glycosylated GP92–101 sequence at the surface of infected cells. However, from our study and others using artificially glycosylated peptides, it is clear that glycopeptides can bind to MHC class I molecules and be immunogenic, indicating that a T cell repertoire exists for specific recognition of class I restricted glycosylated peptides (34 –37). It is thus quite possible that the absence of N-glyco-sylated peptides naturally presented by classical MHC class I molecules may be due to limitations at the level of the antigen processing pathway. For example, in the cytosol, only peptide: N-glycanase-treated glycoproteins but not untreated glycopro-teins are substrates for proteasomal degradation and antigen processing in the ER is blocked as long as glycosylation is not pharmacologically or genetically inhibited (38). Whether or not O-glycosylated peptides produced in the cytosol can be pre-sented by MHC class I molecules remains to be studied.
An oxidized form of the non-glycosylated peptide was also present in our extract. Even though the exact nature of this
FIG. 7. Effect of Endo H treatment of peptide eluate on recognition by anti-(GlcNac)-N95-specific CTL. Ma-terial acid-eluted from the surface of LCMV-infected MC57 cells (13 108cell eq) treated (solid circles) or not (open
cir-cles) with Endo H as described under
“Materials and Methods” was fraction-ated on RP-HPLC using gradient system I, and the fractions were tested for their ability to sensitize RMA cells to lysis by anti-(GlcNac)-N95 CTL at an E:T ratio of 10:1. Synthetic (GlcNac)-N95 peptide (dashed circles) whose retention time is indicated by the arrow was fractionated, treated, and analyzed under the same ex-perimental conditions.
FIG. 8. Effect of DTT treatment of peptide eluate on recogni-tion by anti-N95-specific CTL. Peptide eluate was treated (solid circles) or not (open circles) with DTT before HPLC fractionation as
described under “Materials and Methods.” Spontaneous chromium re-lease from RMA cells in the presence or absence of DTT-treated blank sample was 27% and 2%, respectively. Anti-N95 CTL was then tested against fractions 20 –50 as described in the legend of Fig. 3.
form remains to be fully characterized, we have shown that it was recognized by anti-N95 CTL but not by anti-95D CTL and that this peptide was absent from DTT-treated samples. It has to be noted that Cys residues can readily be oxidized in biolog-ical fluids or during storage of Cys-containing peptides. While in previous reports such a modification was produced during antigen processing (27, 39), we lack evidence that such a mech-anism occurs in our case.
What may be the physiopathological consequences of proc-essing and presentation of multiple forms of a single antigenic sequence? In terms of MHC presentation, the observation that the high binding affinity of GP92–101 for H-2Dbwas left
un-affected by either N-glycosylation or de-N-glycosylation is con-sistent with previous data showing that position 4 of H-2Db
-restricted peptides tolerates a wide variety of substitutions (20). The side chain of the residue at position 4 points out of the H-2Dbbinding groove and represents a TCR contact residue for
most of the known H-2Db-restricted viral epitopes (40 – 42).
Generation of multiple forms of a peptide that display similar high MHC binding properties but have different TCR activa-tion properties from a single sequence potentially increases the diversity of antigens presented to T cells. Thus, in terms of TCR recognition, N-glycosylation alone or followed by de-N-glycosy-lation can create distinct epitopes. Indeed, while CTL gener-ated against N95 efficiently recognized both N95 and D95 (the latter even being recognized better than the immunogen), CTL generated against D95 selectively recognized this peptide. Im-munization of C57BL/6 mice with N95 induces a protective CTL response against LCMV and splenocytes from LCMV-infected mice can be maintained in culture with N95 pulsed target cells and stained with N95-H-2Db tetramers (22, 26).
Presentation of multiple forms of the same viral antigen should be considered as an advantage for the host in its fight against viral infection. However, the observation that despite the pres-ence of its multiple forms at the surface of LCMV-infected cells, an efficient GP92–101-specific primary CTL response is not observed in LCMV-infected C57BL6 mice against the unmodi-fied N95 form (21, 22) or post-translationally modiunmodi-fied D95 and (GlcNac)-N95 forms (data not shown), demonstrates that the LCMV GP92–101 remains a non-immunodominant epitope. So, conversely, could processing and presentation of multiple forms of LCMV GP92–101 be a cause of its non-immunodominance? The observation that the protective capacity of CTL is influ-enced by the diversity of viral peptides generated within in-fected cells (43) would support this latter hypothesis. Another attractive and elegant hypothesis is that non-immunodomi-nance may result from TCR antagonism naturally generated by multiple antigen processing from the same sequence. Both we and others have shown that altered peptide ligands resulting from mutation of an antigenic peptide can differentially affect TCR recognition, provoking partial agonism or antagonism (44 – 46) and/or allowing viruses to evade the CTL response (41). Further, it has recently been shown that antigen process-ing could generate both stimulatory and antagonist peptides from a single class II model epitope, which may have important implications for T cell immunoregulation (47). These different hypotheses are currently under investigation in our laboratory. Acknowledgments—We thank Persephone Borrow and Marilyn
Magazin for critical reading of the manuscript.
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