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ík*
Wistar Institute, Philadelphia, Pennsylavania 19104; and Jefferson Cancer Institute, Thomas Jefferson University, Philadelphia, Pennsylvania 19107-6731
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Class I molecules of the MHC function to display peptides from intracellular proteins on the cell surface for inspection by CD8+ T cells (TCD8+). In most cases, peptides derive from a cytosolic/nuclear pool of proteins through the action of proteasomes or other cytosolic proteases (1), and are delivered to class I molecules by transporter associated with antigen presentation (TAP)1, the MHC-encoded peptide transporter (2). Newly synthesized
Although many class I binding peptides derive from cytosolic or nuclear proteins, proteins targeted to the ER (perhaps a third of all cellular proteins, and important antigens for membrane viruses) also represent a major source of peptides. Most peptides from ER-targeted proteins are presented in a TAP-dependent manner. This, in conjunction with earlier findings that removal of the ER-insertion sequence from integral membrane proteins increases their antigenicity (6, 7), indicates that antigenic peptides from ER-targeted proteins are often produced by cytosolic proteases. It remains unresolved however, to what extent this reflects generation of peptides from defective cytosolic forms of the protein that are not exported to the ER, as originally proposed (8), versus reimportation of exported proteins to the cytosol where they are processed as cytosolic antigens. The latter possibility has gained credence with recent findings from several systems that membrane-anchored and soluble proteins and peptides can be returned to the cytosol where they are degraded in a proteasome dependent manner (9–12).
In the present study we examine this question using Asn-linked glycosylation (commonly termed N-linked glycosylation) as a means of determining whether the source protein has visited the ER. N-linked glycosylation occurs exclusively in the ER, where Glc3Man9GlcNac2 is added to Asn residues present in the sequence Asn X Ser/Thr (X, any residue except Pro). Under normal circumstances, glycosylation is believed to occur cotranslationally as proteins are threaded through the translocon. Extrusion through the translocon is not an absolute requirement, however, since synthetic peptides can also be glycosylated when delivered to the ER via TAP (13–16). Protein conformation can influence glycosylation, since potential glycosylation sites present on exported proteins are not always used.
Glycosylation of class I binding peptides is likely to modify or destroy peptide antigenicity. Depending on the orientation of the glycosylated Asn in the class I binding groove, addition of the oligosaccharide (which is bulkier than the typical class I binding peptide) would be expected to prevent either class I binding or T cell recognition. Indeed, the addition of N-linked oligosaccharides to MHC class II–associated peptides has been found to abolish presentation to TCD4+ (17–19). More directly, addition of O- or N-linked oligosaccharides to MHC class I–associated peptides either greatly reduces binding to class I molecules or interferes with recognition by TCD8+, depending on the orientation of the modified residue (20). N-linked oligosaccharides can be removed by peptide N glycanase (PNGase); this results in the conversion of the oligosaccharide modified residue from Asn to Asp. Skipper et al. found just such a conversion in a peptide derived from an ER-targeted protein (21). Although the TAP dependence of peptide presentation was not reported in this study, it is thought that PNGase is located exclusively in the cytosol/ nucleus (22), which, if true, would mean that peptides were derived from glycosylated protein returned to the cytosol. In the present study, we provide evidence that this ER to cytosol pathway does indeed contribute to the generation of antigenic peptides from proteins cotranslationally targeted to the secretory pathway.
Mice and Cytotoxicity Assays.
Experimental release – medium release/ total release – medium release x 100.
Synthetic Peptides.
Biochemical Procedures.
Obviously, for this to be a useful strategy, the amino acid substitution alone cannot adversely affect the binding of the peptides to Kd. This was examined in two ways. First, RMA/S Kd cells incubated at 26°C to accumulate cell-surface peptide–receptive molecules were exposed to decreasing amounts of peptides for an hour at 26°C, and then for 2 h at 37°C to denature class I molecules lacking peptides. BFA was included with peptides to prevent the transport of additional Kd molecules to the cell surface. Conformed class I molecules were detected using the mAb SF1.1.1 conjugated to FITC. The peptide concentration required to preserve half of the cell-surface Kd molecules provides an approximation of peptide affinity for Kd. This revealed that the Asn149- and Asn149Ala151-containing peptides have Kas approximately 2.5–3-fold higher than the wild-type peptide (Ka = 1/[peptide concentration], yielding values for variant and wild-type peptides, respectively, of 1–1.2 x 107 M–1 versus 4.2 x 106 M–1) (Fig. 1). By contrast, a peptide containing Asp149 (which would result from PNGase-mediated removal of the N-linked oligosaccharide from Asn149) was bound with an estimated Ka 230-fold lower than the wild-type peptide (1.8 x 104 M–1). 
-β2microglobulin heterodimers associate with TAP and remain tethered until released by a conformational alteration induced by peptide binding (3, 4). These events occur in the endoplasmic reticulum (ER) (5).
Materials and Methods
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Abstract
Materials and Methods
Results
Discussion
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Recombinant Vaccinia Viruses.
Recombinant vaccinia viruses (rVVs) expressing Kd, TAP (1 and 2), and influenza virus nucleoprotein (NP) have been described (23, 24). A rVV expressing ICP47 (25) was provided by B. Rouse (University of Tennessee, Knoxville, TN). The full-length NP gene modified by standard molecular genetic methods to encode molecules listed in Table 1 was inserted into the thymidine kinase locus of vaccinia viruses (VVs) by homologous recombination as described using the pSC11 plasmid to express foreign proteins under the control of the VV p7.5 early/late promoter (26). NP was directed to the secretory pathway using the signal sequence from β interferon (27). The NP-coding sequences of all of the rVVs were verified by sequencing PCR-amplified copies of full-length NP genes isolated from the rVV.
6–8-wk-old BALB/cByJ (H-2d) and CBA/J (H-2k) mice were obtained from Jackson Labs (Bar Harbor, ME). Mice were immunized with PR8 by intraperitoneal injection, and with rVV by intravenous injection. Target cells were infected with viruses as described previously (28). To generate TCD8+, splenocytes derived from animals immunized with viruses 2–6 wk previously were stimulated in vitro for 7 d with either antigenic peptides at 1 µg/ml or virus-infected autologous splenocytes as described (29). In some cases, TCD8+ lines were used in place of secondary cultures. Lines were propagated by weekly stimulation with L929-Kd cells that had been incubated for 3 h at 37°C with IMDM supplemented with 20 µg/ml human β2-microglobulin (Sigma Chemical Co., St. Louis, MO) and 1.2 µg/ml synthetic peptide, fixed for 10 min at room temperature with 0.5% paraformaldehyde in PBS, and washed by three consecutive 30-min incubations with IMDM at 37°C. 1–2 d after stimulation with cells, fresh IMDM supplemented with 10% (vol/vol) supernatant from rat splenocytes stimulated with Con A was added. This stimulation protocol was repeated weekly. Microcytotoxicity assays were performed as previously described (28). Where indicated, brefeldin A (BFA; Sigma Chemical Co.) was added to cells at a concentration of 5 µg/ml before assay and at a concentration of 2.5 µg/ml during the cytotoxicity assay to block further antigen presentation (29). Data are expressed as percent-specific release defined as:
Synthetic peptides were provided by the Biological Resources Branch (National Institute of Allergy and Infectious Diseases, Bethesda, MD). Glycopeptides were produced as described (18). All peptides were purified by HPLC and analyzed by mass spectroscopy to confirm purity and mass.
2 x 10 7 L929 cells infected for 1 h with VV were incubated in 2 ml Met-free DMEM for 30 min at 37°C. After adding 200 µCi [35S]methionine in 200 µl Met-free DMEM, cells were incubated for an additional 10 min at 37°C. After washing with PBS, cells were incubated for 20 min at 0°C with 0.14 M NaCl, 1 mM EDTA, 1% Triton X-100, 1 mM PMSF, and 10 mM Tris HCl, pH 7.4, and the nuclei were pelletted by centrifugation at 15,000 g. Supernatants were incubated with protein A–Sepharose preloaded with the anti-NP mAb H16-L10. After extensive washing, beads were boiled in sample buffer according to Laemmli (30), or in 0.1 M sodium acetate, 3 mM EDTA, and 0.25% SDS, pH 6. In the latter case, samples were split in half, incubated overnight at 37°C with or without 2 mU endo-β-N-acetylglucosaminidase (endo H). All samples were analyzed by SDS-PAGE according to Laemmli. After fixing, gels were dried under vacuum and exposed to Phosphorimager screens (Molecular Dynamics, Sunnyvale, CA), which were imaged using the associated hardware and software. For quantitation of viral gene expression in cells used for the microcytoxicity assay, an aliquot of 106 cells was removed, incubated in methionine-deficient medium for 15 min, and labeled with [35S]-methionine for 45 s. Labeling was terminated by the addition of Laemmli sample buffer heated to 100°C. Samples were analyzed by SDS-PAGE, and the amount of protein in fixed gels determined using a Phosphorimager (Molecular Dynamics). Images of gels were arranged and labeled with Adobe Photoshop (Adobe Systems, Mountain View, CA) and printed with a Fujix 3000 pictography digital printer (Fuji Photo Film USA, Elmsford, NY).
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Abstract
Materials and Methods
Results
Discussion
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Experimental Strategy and Characterization of Antigenic Peptides.
As a model antigen we used influenza virus NP synthesized by rVV. NP is synthesized on free ribosomes in the cytosol and efficiently translocated to the nucleus due to a nuclear localization sequence. In H-2d mice, the TCD8+ response to NP focuses on an immunodominant Kd-restricted peptide located at residues 147–155 (TYQRTRALV). Conversion of the Gln149 to Asn creates a N-linked glycosylation site. To study the effects of glycosylation on antigen processing, we produced rVVs expressing NP with Gln149 or Asn149 directed to the ER by appendage of a NH2-terminal ER insertion sequence (termed secreted NP [SNP]; Table 1). We controlled for effects of the Gln149
Asn substitution on peptide liberation, class I binding, and TCR recognition by using rVVs expressing the same proteins in the cytosol. To examine whether any alterations in presentation of SNPN are related to glycosylation, we produced rVVs expressing cytosolic or ER-targeted NPs in which the glycosylation site created by the Gln149 Asn substitution is destroyed by converting Thr151 to Ala (Table 1).
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In the experiments that follow, we used TCD8+ populations or lines raised to the wild-type sequence as a measure of presentation of the 147–155 determinant from the different cytosolic or ER-targeted NPs. The effects of the amino acid substitutions on peptide antigenicity varied somewhat with the TCD8+ population or line used. In two of the experiments described below, the Asn149- and Asn149Ala151-containing peptides sensitized target cells for lysis by NP-specific TCD8+ with similar or slightly greater efficiencies than the wild-type peptide (see Figs. 3 and 5). The Asp149-containing peptide routinely required 100–1,000 times the concentration of Gln149- or Asn149-containing peptides to achieve a similar level of lysis (not shown).
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Characterization of SNP.
The function of the ER-targeting sequence was confirmed by SDS-PAGE analysis of total cell extracts of rVV-infected cells metabolically labeled with [35S]methionine. As seen in Fig. 2 A, the secreted forms of NP migrated more slowly than the cytosolic/nuclear forms. SNP and SNPNRA comigrated, whereas SNPN migrated more slowly, which is consistent with the existence of one site for N-linked glycosylation in SNP and SNPNRA (NP has a natural glycosylation site at position 21) and two sites in SNPN. This was confirmed by digestion of immunocollected SNP and SNPN with endo H, which eradicated the difference in mobility between the ER-targeted and nontargeted forms of NP (Fig. 2 B). It is important to note that all three forms of SNP were efficiently delivered to the ER and glycosylated at all potential sites, since we did not detect, by either immunoprecipitation or by Western blotting (not shown), full-length forms that were nonglycosylated or partially glycosylated. Upon prolonged chasing at 37°C, all three forms of SNP became endo H–resistant and were secreted into the medium with a t1/2 of 
3 h (not shown).
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Together, these findings demonstrate that (a) SNPs are efficiently targeted to the ER, (b) all potential glycosylation sites are used in SNPs to within the limits of detection, and (c) a minor portion of SNPs make their way to the nucleus.
Effect of Gln149 Asn Substitution on NP/SNP Antigenicity and Immunogenicity.
The efficiency of generating the NP147-155 peptide from the six different forms of NP was determined using a NP-specific TCD8+ line that recognizes the wild type, Asn149- and Asn149Ala151-containing peptides with similar efficiency (Fig. 3, inset). The efficiency of peptide generation was estimated by adding BFA to L-Kd cells at increasing intervals after infection with rVVs (Fig. 3; 32). As with other proteins, targeting NP to the ER reduced the efficiency of peptide generation. Modification of amino acids 149 or 151 had little effect on presentation of cytosolic NP. The same alterations, however, had clear effects on presentation of SNP. The Gln149 Asn substitution significantly reduced presentation of SNP. The similar levels of presentation of SNPNRA and SNP indicates that glycosylation of Asn149 is responsible for decreased presentation of SNPN relative to SNP.
To eliminate the possibility that the differences in antigen presentation reflected differences in levels of expression of the various antigens (and not real differences in antigen processing), we quantitated the amounts of NP produced by the target cells used in this experiment. Cells were radiolabeled with [35S]methionine for 45 s and immediately immersed in boiling SDS-PAGE sample buffer. This method allows recovery of virtually all of the biosynthesized proteins, and not just those extracted into relatively mild detergents that are capable of binding antibody (the technique most commonly used to quantitate antigen expression). SDS-PAGE of extracts revealed that newly synthesized NP could be easily detected due to the strength of the VV p7.5 promoter combined with the ability of VV to shut down host cell protein synthesis. As seen in Fig. 2 A, the difference in presentation between the secreted forms of NP cannot be attributed to differences in levels of expression. Indeed, increasing the amount of SNPN synthesized by increasing the virus dose by threefold (SNPNHigh) did not increase presentation to TCD8+ (Fig. 3). NP-specific TCD8+ from H-2k mice recognize residues 50–57 in association with Kk (33). Unlike the Kd-restricted determinant, the presentation of NP50-57 was not adversely affected by the Gln149 Asn substitution in SNP (Fig. 4). In fact, VV-SNPN–infected cells were recognized at slightly higher levels by Kk-restricted TCD8+ than cells expressing SNP, whereas the very same target cells gave the reverse hierarchy of lysis using Kd-restricted TCD8+. This provides functional evidence that the diminished recognition of VV-SNPN–infected cells relative to VV-SNP–infected cells is not related to diminished levels of SNPN expression. Moreover, this experiment indicates that the inhibitory effect of glycosylation on SNP antigen processing is limited to the determinant containing the glycosylation site.
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Asn substitution into SNP decreased the immunogenicity of the rVV 100-fold relative to the other viruses (in additional experiments we detected priming after inoculation with 106 plaque-forming units/ mouse [not shown]). This effect was specific for the secreted form, since the same substitution actually increased the immunogenicity of cytosolic NP 10-fold. The low immunogenicity of VV-SNPN cannot be attributed to poor infectivity in vivo since it primed for VV-specific responses with similar efficiency as the other rVVs (not shown). These findings demonstrate that the presence of an N-linked glycosylation site in a secreted protein specifically affects its antigenicity and immunogenicity, presumably due to the attachment of an N-linked oligosaccharide in the ER. Creation of the same site in a cytosolic version of the protein has no significant effect on antigenicity.
TAP Dependence of Antigen Presentation.
The involvement of TAP in the presentation of NP, NPN, SNP, and SNPN was examined using T2-Kd cells, a TAP-deficient human lymphoblastoid cell line expressing Kd from a transfected gene (34; Fig. 5 A). None of the corresponding rVVs were able to sensitize T2-Kd cells for lysis by NP-specific TCD8+. The precise hierarchy of presentation of the various gene products observed in L-Kd cells was faithfully restored, however, by coinfection with VV-TAP (1 and 2), which encodes both human TAP subunits (35). Lysis of SNPN-expressing cells was at levels less than half that observed with SNP. This cannot be attributed to less efficient recognition of the Asn149-containing peptide by the TCD8+ line used since NPN was presented slightly more efficiently than NP and the synthetic peptides were recognized with similar efficiency. Nor can it be attributed to decreased expression of SNPN relative to SNP. As in the previous experiment, cells were briefly radiolabeled and the expression of NPs quantitated after SDS-PAGE of whole cell lysates. This demonstrated that SNPN was expressed in similar amounts to SNP (Fig. 5 legend). To eliminate the possibility that a minor population of cells synthesized most of the antigen, NP expression was examined by immunoperoxidase staining of fixed and permeabilized target cells. As seen in Fig. 5, a similar percentage of cells expressed the various forms of NP (SNPN was actually detected in a greater percentage of cells than SNP).
These findings demonstrate that the TAP-dependent presentation of the 147-155 peptide derived from SNP is blocked by glycosylation. TAP has recently been shown to directly bind to class I molecules (3, 4), and its potential as a molecular chaperone in class I folding has been suggested (36). Thus, it is possible that the TAP dependence of presentation of the NP147-155 determinant reflects a role for TAP in facilitating the association of Kd with the free peptide in the ER lumen, and not the transport of 147-155 peptide from the cytosol. To examine this possibility, we used an rVV expressing the herpes simplex virus protein ICP47 to specifically inhibit the transport function of TAP (23, 37– 40). Coexpression of TAP with ICP47 blocked the TAP-mediated rescue of both NP and SNP (Fig. 5 B).
These findings demonstrate that the 147-155 determinant produced from SNP is delivered to class I molecules by TAP from the cytosol, and that this presentation is partially blocked by glycosylation.
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A synthetic version of the Asn149-substituted 147-155 peptide is glycosylated in a TAP-dependent manner when added to cells permeabilized by streptolysin O (our unpublished results). Thus, we interpret the efficient presentation NPN to indicate that the Asn149-containing peptide associates with Kd before the peptide can be glycosylated. This is consistent with the idea that TAP delivers peptides directly to the class I molecules they bind (3, 4). The practical importance of this is that the antigenicity and immunogenicity of peptides in cytosolic/nuclear proteins are probably not affected by the presence of a consensus site for N-linked glycosylation.
By contrast, the antigenicity, and particularly the immunogenicity of SNP, were greatly affected by the presence of a glycosylation site, indicating that glycosylation can have a major negative effect on the antigenicity of peptides in ER-targeted proteins. The disproportionate effect of the Gln149
Asn substitution on SNP immunogenicity relative to its effect on antigenicity indicates that presentation of antigen to primary T cells in vivo is fundamentally different from in vitro presentation. This may reflect differences in antigen processing in the APCs used in vivo versus the various tumor cells we have used. An intriguing possibility that we favor is that in vivo presentation of SNP relies largely on processing of exogenous antigen scavenged from infected cells by professional APCs (41). Exogenous antigens would consist of the metabolically stable proteins produced by cells, and in this case, the very large difference between presentation of SNP and SNPN would be expected based on the high efficiency of glycosylation of Asn149.
The present results do not distinguish in which secretory organelle egress of SNP to the cytosol occurs (anywhere from ER to lysosomes is possible). Cells are able to present exogenous antigens in a TAP-dependent manner, due to release of pinocytosed material to the cytosol. This pathway is largely limited to phagocytosing cells, however. Moreover, in additional experiments (not shown), we found that presentation of SNP could not be attributed to uptake of secreted material, since coincubation of VV-SNP–infected cells with 51Cr-labeled uninfected cells during the course of a 6-h cytotoxicity assay did not result in the lysis of uninfected cells (not shown). It was recently shown that two cytomegalovirus proteins, US2 and US11, have the remarkable ability to transport newly synthesized class I molecules from the ER to the cytosol, probably by inducing reverse translocation through the translocon (10, 11). The authors proposed that cytomegalovirus takes advantage of a constitutive operating cellular pathway that underlies the phenomenon of ER-associated degradation of defective or unassembled proteins. We propose that SNP is reimported from the ER to the cytosol via the same pathway.
The negative effect of glycosylation on peptide antigenicity could have several causes. First, if the oligosaccharide is not removed, peptide antigenicity would be eradicated by interference with TAP-mediated transport, Kd binding, or TCR recognition. Second, removal of the oligosaccharide by cytosolic PNGase would create an Asp149-substituted peptide with low affinity for Kd. In additional experiments, we found that this peptide sensitizes cells 1,000-fold less efficiently than the wild-type peptide for lysis mediated by TCD8+ raised to NP, and that these TCD8+ do not recognize cells expressing rVV encoded cytosolic or ER-targeted Asp149-containing peptides (our unpublished results). Thus, removal of the glycan would destroy peptide antigenicity as surely as its nonremoval.
The inhibitory effect of glycosylation on in vitro or in vivo presentation of SNP was incomplete. Based on the argument presented in the preceding paragraph, the residual presentation of SNPN is almost certainly due to incomplete glycosylation of Asn149. Although we cannot eliminate the possibility that a small amount of SNPN escapes glycosylation at Asn149, the degree of residual presentation of SNPN relative to SNP seems to be inconsistent with our failure to detect SNPN lacking one or both N-linked oligosaccharides by SDS-PAGE analysis of mAb-collected material. Instead, we believe that the residual presentation of SNPN reflects the generation of peptides from defective ribosomal products (DRiPs; 42), misfolded, possibly truncated forms of SNPN that are not inserted into the ER and are processed by the standard cytosolic route.
In conclusion, the present findings expand the possible mechanisms underlying the presentation of secreted and membrane-bound proteins. It will be important to test in future experiments whether the results we describe are limited to cytosolic proteins artificially routed to the secretory pathway, or also apply to naturally exported proteins that have evolved to be resistant to secretory proteases. The major mode of peptide generation from any given exported protein must be determined on a case by case basis until general principles have been more firmly established.
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Acknowledgments |
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Submitted: 9 April 1997
Revised: 13 June 1997
1 Abbreviations used in this paper: BFA, brefeldin A; endo H, endo-β-N-acetylglucosaminidase; ER, endoplasmic reticulum; NP, nucleoprotein; PNGase, peptide N glycanase; rVV, recombinant VV; TAP, transporter associated with antigen presentation; VV, vaccinia virus.
I. Baík, H.L.Snyder, and L.C. Antón contributed equally to this work, and are listed in random order.
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1 York I & Rock KL. Antigen processing and presentation by the class I major histocompatibility complex, Annu Rev Immunol, 1996, 14, 369–396.[Medline]
2 Heemels M-T & Ploegh H. Generation, translocation, and presentation of MHC class-I restricted peptides, Annu Rev Biochem, 1995, 64, 463–491.[Medline]
3 Suh W-K, Cohen-Doyle MF, Fruh K, Wang K, Peterson PA & Williams DB. Interaction of MHC class I molecules with the transporter associated with antigen processing, Science (Wash DC), 1994, 264, 1322–1326.
4 Ortmann B, Androlewicz MJ & Cresswell P. MHC class I/β2-microglobulin complexes associate with TAP transporters before peptide binding, Nature (Lond), 1994, 368, 864–867.[Medline]
5 Lapham CK, Bacik I, Yewdell JW, Kane KP & Bennink JR. Class I molecules retained in the endoplasmic reticulum bind antigenic peptides, J Exp Med, 1993, 177, 1633–1641.
6 Townsend ARM, Bastin J, Gould K & Brownlee GG. Cytotoxic T lymphocytes recognize influenza hemagglutinin that lacks a signal sequence, Nature (Lond), 1986, 324, 575–577.[Medline]
7 Townsend A, Bastin J, Gould K, Brownlee G, Andrew M, Coupar B, Boyle D, Chan S & Smith G. Defective presentation to class I–restricted cytotoxic T lymphocytes in vaccinia-infected cells is overcome by enhanced degradation of antigen, J Exp Med, 1988, 168, 1211–1224.
8 Hammond SA, Johnson RP, Kalams SA, Walker BD, Takiguchi M, Safrit JT, Koup RA & Siliciano RF. An epitope-selective, transporter associated with antigen presentation (TAP)-1/2–independent pathway and a more general TAP-1/2–dependent antigen-processing pathway allow recognition of the HIV-1 envelope glycoprotein by CD8+CTL, J Immunol, 1995, 154, 6140–6156.[Abstract]
9 Roelse J, Grommé M, Momburg F, Hämmerling G & Neefjes J. Trimming of TAP-translocated peptides in the endoplasmic reticulum and in the cytosol during recycling, J Exp Med, 1994, 180, 1591–1597.
10 Wiertz EJH, Jones TR, Sun L, Bogyo M, Geuze HJ & Ploegh HL. The human cytomegalovirus US11 gene product dislocates MHC class I heavy chains from the endoplasmic reticulum of the cytosol, Cell, 1996, 84, 769–779.[Medline]
11 Wiertz EJHJ, Tortorella D, Bogyo M, Yu J, Mothes W, Jones TR, Rapoport TA & Ploegh HL. Sec61-mediated transfer of a membrane protein from the endoplasmic reticulum to the proteosome for destruction, Nature (Lond), 1996, 384, 432–438.[Medline]
12 McCracken AA & Brodsky JF. Assembly of ER-associated protein degradation in vitro: dependence on cytosol, calnexin and ATP, J Cell Biol, 1996, 132, 291–298.
13 Schumacher TNM, Kantesaria DV, Heemels M-T, Ashton-Rickardt PG, Shepherd JC, Fruh K, Yang Y, Peterson PA, Tonegawa S & Ploegh HL. Peptide length and sequence specificity of the mouse TAP1/TAP2 translocator, J Exp Med, 1994, 179, 533–540.
14 Neefjes JJ, Momburg F & Hämmerling GJ. Selective and ATP-dependent translocation of peptides by the MHC-encoded transporter, Science (Wash DC), 1993, 261, 769–771.
15 van Endert PM, Tampé R, Meyer TH, Tisch R, Bach J-F & McDevitt HO. A sequential model for peptide binding and transport by the transporters associated with antigen processing, Immunity, 1994, 1, 491–500.[Medline]
16 Androlewicz MJ & Cresswell P. Human transporters associated with antigen processing possess a promiscuous peptide-binding site, Immunity, 1994, 1, 7–14.[Medline]
17 Thomas DB, Hodgson J, Riska PF & Graham CM. The role of the endoplasmic reticulum in antigen processing: N-glycosylation of influenza hemagglutininin abrogates CD4+cytotoxic T cell recognition of endogenously processed antigen, J Immunol, 1990, 144, 2789–2794.[Abstract]
18 Jackson DC, Drummer HE, Urge L, Otvos L Jr & Brown LE. Glycosylation of a synthetic peptide representing a T-cell determinant of influenza virus hemagglutinin results in loss of recognition by CD4+T-cell clones, Virology, 1994, 199, 422–430.[Medline]
19 Ishioka G, Lamont A, Thomson D, Bulbow N, Gaeta F, Sette A & Grey H. MHC interaction and T cell recognition of carbohydrates and glycopeptides, J Immunol, 1992, 148, 2446–2451.[Abstract]
20 Haurum JS, Arsequell G, Lellouch AC, Wong SYC, Dwek RA, McMichael AJ & Elliot T. Recognition of carbohydrate by major histocompatibility complex class I–restricted, glycopeptide-specific cytotoxic T lymphocytes, J Exp Med, 1994, 180, 739–744.
21 Skipper JCA, Hendrickson RC, Gulden PH, Brichard V, Van Pel A, Chen Y, Shabanowitz J, Wolfel T, Slingluff CL, Boon T et al.. An HLA-A2–restricted tyrosinase antigen on melanoma cells results from posttranslational modification and suggests a novel pathway for processing of membrane proteins, J Exp Med, 1996, 183, 527–534.
22 Suzuki T, Seko A, Kitajima K, Inoue Y & Inoue S. Purification and enzymatic properties of peptide: N-glycanase from C3H mouse–derived L-929 fibroblast cells, J Biol Chem, 1994, 269, 17611–17618.
23 Hill A, Jugovic P, York I, Russ G, Bennink J, Yewdell J, Ploegh H & Johnson D. Herpes simplex virus turns off the TAP to evade host immunity, Nature (Lond), 1995, 375, 411–414.[Medline]
24 Eisenlohr LC, Bacik I, Bennink JR, Bernstein K & Yewdell JW. Expression of a membrane protease enhances presentation of endogenous antigens to MHC class I–restricted T lymphocytes, Cell, 1992, 71, 963–972.[Medline]
25 Banks TA, Jenkins FJ, Kanangat S, Nair S, Dasgupta S, Foster CM & Rouse BT. Vaccination with the immediate-early protein ICP47 of herpes simplex virus–type 1 (HSV-1) induces virus-specific lymphoproliferation, but fails to protect against lethal challenge, Virology, 1994, 200, 236–245.[Medline]
26 Chakrabarti S, Brechling K & Moss B. Vaccinia virus expression vector: coexpression of β-galactosidase provides visual screening of recombinant virus plaques, Mol Cell Biol, 1985, 5, 3403–3409.
27 Derynck R, Content J, DeClercq E, Volckaert G, Tavernier J, Devos R & Fiers W. Isolation and structure of a human fibroblast interferon gene, Nature (Lond), 1980, 285, 542–547.[Medline]
28 Snyder HL, Yewdell JW & Bennink JR. Trimming of antigenic peptides in an early secretory compartment, J Exp Med, 1994, 180, 2389–2394.
29 Yewdell JW & Bennink JR. Brefeldin A specifically inhibits presentation of protein antigens to cytotoxic T lymphocytes, Science (Wash DC), 1989, 244, 1072–1075.
30 Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4, Nature (Lond), 1970, 227, 680–685.[Medline]
31 Kornfeld R & Kornfeld S. Assembly of asparagine-linked oligosaccharides, Annu Rev Biochem, 1985, 54, 631–664.[Medline]
32 Esquivel F, Yewdell JW & Bennink JR. RMA/S cells present endogenously synthesized cytosolic proteins to class I–restricted cytotoxic T lymphocytes, J Exp Med, 1992, 175, 163–168.
33 Cossins J, Gould K & Brownlee GG. Peptides shorter than a minimal CTL epitope may have a higher binding affinity than the epitope for the class I Kkmolecule, Virology, 1993, 195, 851–854.[Medline]
34 Salter RD & Cresswell P. Impaired assembly and transport of HLA-A and -B antigens in a mutant TxB cell hybrid, EMBO (Eur Mol Biol Organ) J, 1986, 5, 943–949.[Medline]
35 Russ G, Esquivel F, Cresswell P, Spies T, Yewdell JW & Bennink JR. Assembly, intracellular localization, and nucleotide binding properties of the human peptide transporters Tap1 and Tap2 expressed by recombinant vaccinia viruses, J Biol Chem, 1995, 270, 21312–21318.
36 Day PM, Esquivel F, Lukszo J, Bennink JR & Yewdell JW. Effect of TAP on the generation and intracellular trafficking of peptide-receptive major histocompatibility complex class I molecules, Immunity, 1995, 2, 137–147.[Medline]
37 York IA, Roop C, Andrew DW, Riddell SR, Graham FL & Johnson DC. A cytosolic herpes simplex virus protein inhibits antigen presentation to CD8+ T lymphocytes, Cell, 1994, 77, 525–535.[Medline]
38 Früh K, Ahn K, Djaballah H, Sempé P, van Endert PM, Tampé R, Peterson PA & Yang Y. A viral inhibitor of peptide transporters for antigen presentation, Nature (Lond), 1995, 375, 415–418.[Medline]
39 Ahn K, Meyer TH, Uebel S, Sempé P, Djaballah H, Yang Y, Peterson PA, Früh K & Tampé R. Molecular mechanism and species specificity of TAP inhibition by herpes simplex virus protein ICP47, EMBO (Eur Mol Biol Organ) J, 1996, 15, 3247–3255.[Medline]
40 Tomazin R, Hill AB, Jugovic P, York I, van Endert P, Ploegh HL, Andrews DW & Johnson DC. Stable binding of the herpes simplex virus ICP47 protein to the peptide binding site of TAP, EMBO (Eur Mol Biol Organ) J, 1996, 15, 3256–3266.[Medline]
41 Bevan MJ. Antigen presentation to cytotoxic T lymphocytes in vivo, J Exp Med, 1995, 182, 639–641.
42 Yewdell JW, Antón LC & Bennink JR. Defective ribosomal products (DRiPs). A major source of antigenic peptides for MHC class I molecules? , J Immunol, 1996, 157, 1823–1826.[Abstract]
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