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Introduction of a glycosylation site into a secreted protein provides evidence for an alternative antigen processing pathway: transport of precursors of major histocompatibility complex class I-restricted peptides from the endoplasmic reticulum to the cytosol.

Bacik I, Snyder HL, Antón LC, Russ G, Chen W, Bennink JR, Urge L, Otvos L, Dudkowska B, Eisenlohr L, Yewdell JW - J. Exp. Med. (1997)

Bottom Line: Introducing an N-linked glycosylation site into the determinant selectively reduced presentation of SNP.This indicates that glycosylation does not interfere with TAP-transported peptides, and therefore that cytosolic peptides derived from SNP must have been exposed to the glycosylation machinery of the endoplasmic reticulum (ER) before their existence in the cytosol.Based on these findings, we propose that TAP-dependent processing of at least some ER-targeted proteins entails the reimportation of protein from the secretory pathway to the cytosol, where the protein is processed via the classical pathway.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, Bethesda, Maryland 20892-0440, USA.

ABSTRACT
We found that the presentation of a H-2Kd-restricted determinant from influenza virus nucleoprotein (NP) to T cells is strictly dependent on expression of the transporter associated with antigen presentation (TAP), regardless of whether NP is expressed as a cytosolic or secreted NP (SNP). Introducing an N-linked glycosylation site into the determinant selectively reduced presentation of SNP. This indicates that glycosylation does not interfere with TAP-transported peptides, and therefore that cytosolic peptides derived from SNP must have been exposed to the glycosylation machinery of the endoplasmic reticulum (ER) before their existence in the cytosol. Based on these findings, we propose that TAP-dependent processing of at least some ER-targeted proteins entails the reimportation of protein from the secretory pathway to the cytosol, where the protein is processed via the classical pathway.

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Antigenicity and  immunogenicity of rVVs expressing various forms of NP.  (A) L-Kd cells were infected for  the indicated times with the  rVVs indicated before BFA was  added and then maintained continuously. After 51Cr labeling,  cells were incubated for 6 h at an  E/T of 5:1 with a TCD8+ line produced by PR8 in vitro secondary  restimulation of splenocytes derived from mice inoculated with  VV-NP. The recognition of synthetic peptides incubated with cells  at the indicated concentration is  shown in the inset. (B) Splenocytes from BALB/c mice immunized with the indicated rVV 3 wk previously were restimulated in vitro for 7 d with the  homologous synthetic peptide (TYQRTRALV or TYNRTRALV), and tested for their ability to lyse P815 cells sensitized with the same peptide. Cultures were used at the same dilution. All values have been corrected for background lysis values obtained with cells in the absence of added synthetic peptides.
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Figure 3: Antigenicity and immunogenicity of rVVs expressing various forms of NP. (A) L-Kd cells were infected for the indicated times with the rVVs indicated before BFA was added and then maintained continuously. After 51Cr labeling, cells were incubated for 6 h at an E/T of 5:1 with a TCD8+ line produced by PR8 in vitro secondary restimulation of splenocytes derived from mice inoculated with VV-NP. The recognition of synthetic peptides incubated with cells at the indicated concentration is shown in the inset. (B) Splenocytes from BALB/c mice immunized with the indicated rVV 3 wk previously were restimulated in vitro for 7 d with the homologous synthetic peptide (TYQRTRALV or TYNRTRALV), and tested for their ability to lyse P815 cells sensitized with the same peptide. Cultures were used at the same dilution. All values have been corrected for background lysis values obtained with cells in the absence of added synthetic peptides.

Mentions: 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).


Introduction of a glycosylation site into a secreted protein provides evidence for an alternative antigen processing pathway: transport of precursors of major histocompatibility complex class I-restricted peptides from the endoplasmic reticulum to the cytosol.

Bacik I, Snyder HL, Antón LC, Russ G, Chen W, Bennink JR, Urge L, Otvos L, Dudkowska B, Eisenlohr L, Yewdell JW - J. Exp. Med. (1997)

Antigenicity and  immunogenicity of rVVs expressing various forms of NP.  (A) L-Kd cells were infected for  the indicated times with the  rVVs indicated before BFA was  added and then maintained continuously. After 51Cr labeling,  cells were incubated for 6 h at an  E/T of 5:1 with a TCD8+ line produced by PR8 in vitro secondary  restimulation of splenocytes derived from mice inoculated with  VV-NP. The recognition of synthetic peptides incubated with cells  at the indicated concentration is  shown in the inset. (B) Splenocytes from BALB/c mice immunized with the indicated rVV 3 wk previously were restimulated in vitro for 7 d with the  homologous synthetic peptide (TYQRTRALV or TYNRTRALV), and tested for their ability to lyse P815 cells sensitized with the same peptide. Cultures were used at the same dilution. All values have been corrected for background lysis values obtained with cells in the absence of added synthetic peptides.
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Related In: Results  -  Collection

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Figure 3: Antigenicity and immunogenicity of rVVs expressing various forms of NP. (A) L-Kd cells were infected for the indicated times with the rVVs indicated before BFA was added and then maintained continuously. After 51Cr labeling, cells were incubated for 6 h at an E/T of 5:1 with a TCD8+ line produced by PR8 in vitro secondary restimulation of splenocytes derived from mice inoculated with VV-NP. The recognition of synthetic peptides incubated with cells at the indicated concentration is shown in the inset. (B) Splenocytes from BALB/c mice immunized with the indicated rVV 3 wk previously were restimulated in vitro for 7 d with the homologous synthetic peptide (TYQRTRALV or TYNRTRALV), and tested for their ability to lyse P815 cells sensitized with the same peptide. Cultures were used at the same dilution. All values have been corrected for background lysis values obtained with cells in the absence of added synthetic peptides.
Mentions: 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).

Bottom Line: Introducing an N-linked glycosylation site into the determinant selectively reduced presentation of SNP.This indicates that glycosylation does not interfere with TAP-transported peptides, and therefore that cytosolic peptides derived from SNP must have been exposed to the glycosylation machinery of the endoplasmic reticulum (ER) before their existence in the cytosol.Based on these findings, we propose that TAP-dependent processing of at least some ER-targeted proteins entails the reimportation of protein from the secretory pathway to the cytosol, where the protein is processed via the classical pathway.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, Bethesda, Maryland 20892-0440, USA.

ABSTRACT
We found that the presentation of a H-2Kd-restricted determinant from influenza virus nucleoprotein (NP) to T cells is strictly dependent on expression of the transporter associated with antigen presentation (TAP), regardless of whether NP is expressed as a cytosolic or secreted NP (SNP). Introducing an N-linked glycosylation site into the determinant selectively reduced presentation of SNP. This indicates that glycosylation does not interfere with TAP-transported peptides, and therefore that cytosolic peptides derived from SNP must have been exposed to the glycosylation machinery of the endoplasmic reticulum (ER) before their existence in the cytosol. Based on these findings, we propose that TAP-dependent processing of at least some ER-targeted proteins entails the reimportation of protein from the secretory pathway to the cytosol, where the protein is processed via the classical pathway.

Show MeSH
Related in: MedlinePlus