Limits...
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.

Show MeSH

Related in: MedlinePlus

TAP dependence  of antigen presentation. (A) T2  Kd cells coinfected with VV-TAP (1 and 2) and the rVVs indicated, or just with the rVV indicated (“No TAP”) were tested  for lysis by a NP-specific TCD8+  line at the E/T ratios indicated.  The recognition of synthetic  peptides incubated with cells at  the indicated concentration by  the same TCD8+ line is shown in  the inset. The amount of antigen  expressed by the target cells 4 h  after infection was determined  by quantitating bands detected in  SDS-PAGE of whole cell extracts as described in Fig. 2. The  adjusted amounts of various  forms of NP synthesized relative  to NP (= 1.00) are: NPN, 1.49;  SNP, 1.01; and SNPN, 1.01. (B)  Effect of ICP47 on antigen presentation. T2 Kd cells infected  with the rVVs indicated were  tested for lysis by the TCD8+ line  used in A at the indicated E/T  ratio. (C) The target cells used in  A were incubated in flat-bottom  96-well plates coated with Con  A to adhere the cells. At the conclusion of the 51Cr release assay,  cells were fixed by 2-min incubation with acetone/methanol  (1:1) and indirectly immunoperoxidase stained using the H16-L10 mAb. Video images of the stained cells were printed using a printer (UP5500; Sony, San Jose, CA), digitized by a flat bed scanner, assembled using  Adobe Photoshop software, and, printed with a digital printer (3000; Fuji Photo Film USA).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2199039&req=5

Figure 5: TAP dependence of antigen presentation. (A) T2 Kd cells coinfected with VV-TAP (1 and 2) and the rVVs indicated, or just with the rVV indicated (“No TAP”) were tested for lysis by a NP-specific TCD8+ line at the E/T ratios indicated. The recognition of synthetic peptides incubated with cells at the indicated concentration by the same TCD8+ line is shown in the inset. The amount of antigen expressed by the target cells 4 h after infection was determined by quantitating bands detected in SDS-PAGE of whole cell extracts as described in Fig. 2. The adjusted amounts of various forms of NP synthesized relative to NP (= 1.00) are: NPN, 1.49; SNP, 1.01; and SNPN, 1.01. (B) Effect of ICP47 on antigen presentation. T2 Kd cells infected with the rVVs indicated were tested for lysis by the TCD8+ line used in A at the indicated E/T ratio. (C) The target cells used in A were incubated in flat-bottom 96-well plates coated with Con A to adhere the cells. At the conclusion of the 51Cr release assay, cells were fixed by 2-min incubation with acetone/methanol (1:1) and indirectly immunoperoxidase stained using the H16-L10 mAb. Video images of the stained cells were printed using a printer (UP5500; Sony, San Jose, CA), digitized by a flat bed scanner, assembled using Adobe Photoshop software, and, printed with a digital printer (3000; Fuji Photo Film USA).

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)

TAP dependence  of antigen presentation. (A) T2  Kd cells coinfected with VV-TAP (1 and 2) and the rVVs indicated, or just with the rVV indicated (“No TAP”) were tested  for lysis by a NP-specific TCD8+  line at the E/T ratios indicated.  The recognition of synthetic  peptides incubated with cells at  the indicated concentration by  the same TCD8+ line is shown in  the inset. The amount of antigen  expressed by the target cells 4 h  after infection was determined  by quantitating bands detected in  SDS-PAGE of whole cell extracts as described in Fig. 2. The  adjusted amounts of various  forms of NP synthesized relative  to NP (= 1.00) are: NPN, 1.49;  SNP, 1.01; and SNPN, 1.01. (B)  Effect of ICP47 on antigen presentation. T2 Kd cells infected  with the rVVs indicated were  tested for lysis by the TCD8+ line  used in A at the indicated E/T  ratio. (C) The target cells used in  A were incubated in flat-bottom  96-well plates coated with Con  A to adhere the cells. At the conclusion of the 51Cr release assay,  cells were fixed by 2-min incubation with acetone/methanol  (1:1) and indirectly immunoperoxidase stained using the H16-L10 mAb. Video images of the stained cells were printed using a printer (UP5500; Sony, San Jose, CA), digitized by a flat bed scanner, assembled using  Adobe Photoshop software, and, printed with a digital printer (3000; Fuji Photo Film USA).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2199039&req=5

Figure 5: TAP dependence of antigen presentation. (A) T2 Kd cells coinfected with VV-TAP (1 and 2) and the rVVs indicated, or just with the rVV indicated (“No TAP”) were tested for lysis by a NP-specific TCD8+ line at the E/T ratios indicated. The recognition of synthetic peptides incubated with cells at the indicated concentration by the same TCD8+ line is shown in the inset. The amount of antigen expressed by the target cells 4 h after infection was determined by quantitating bands detected in SDS-PAGE of whole cell extracts as described in Fig. 2. The adjusted amounts of various forms of NP synthesized relative to NP (= 1.00) are: NPN, 1.49; SNP, 1.01; and SNPN, 1.01. (B) Effect of ICP47 on antigen presentation. T2 Kd cells infected with the rVVs indicated were tested for lysis by the TCD8+ line used in A at the indicated E/T ratio. (C) The target cells used in A were incubated in flat-bottom 96-well plates coated with Con A to adhere the cells. At the conclusion of the 51Cr release assay, cells were fixed by 2-min incubation with acetone/methanol (1:1) and indirectly immunoperoxidase stained using the H16-L10 mAb. Video images of the stained cells were printed using a printer (UP5500; Sony, San Jose, CA), digitized by a flat bed scanner, assembled using Adobe Photoshop software, and, printed with a digital printer (3000; Fuji Photo Film USA).
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