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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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Peptide binding to  Kd. RMA/S Kd cells incubated  overnight at 26°C were incubated for an additional hour with  the indicated concentration of  peptide and BFA (5 μg/ml), and  then at 37°C for 2 h (left). Alternatively, cells were incubated  with 10 μM peptide and BFA,  extensively washed, and then incubated at 37°C for the indicated  time in the absence of peptide  (right). The amount of cell-surface Kd was determined using the  fluorescein-conjugated mAb  SF1.1.1 (PharMingen, San Diego, CA). The concentration of  peptide required for protection  of one half of the Kd molecules  was determined by linear extrapolation of the mean channel fluorescence values.
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Figure 1: Peptide binding to Kd. RMA/S Kd cells incubated overnight at 26°C were incubated for an additional hour with the indicated concentration of peptide and BFA (5 μg/ml), and then at 37°C for 2 h (left). Alternatively, cells were incubated with 10 μM peptide and BFA, extensively washed, and then incubated at 37°C for the indicated time in the absence of peptide (right). The amount of cell-surface Kd was determined using the fluorescein-conjugated mAb SF1.1.1 (PharMingen, San Diego, CA). The concentration of peptide required for protection of one half of the Kd molecules was determined by linear extrapolation of the mean channel fluorescence values.

Mentions: 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 × 107 M−1 versus 4.2 × 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 × 104 M−1).


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)

Peptide binding to  Kd. RMA/S Kd cells incubated  overnight at 26°C were incubated for an additional hour with  the indicated concentration of  peptide and BFA (5 μg/ml), and  then at 37°C for 2 h (left). Alternatively, cells were incubated  with 10 μM peptide and BFA,  extensively washed, and then incubated at 37°C for the indicated  time in the absence of peptide  (right). The amount of cell-surface Kd was determined using the  fluorescein-conjugated mAb  SF1.1.1 (PharMingen, San Diego, CA). The concentration of  peptide required for protection  of one half of the Kd molecules  was determined by linear extrapolation of the mean channel fluorescence values.
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Related In: Results  -  Collection

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

Figure 1: Peptide binding to Kd. RMA/S Kd cells incubated overnight at 26°C were incubated for an additional hour with the indicated concentration of peptide and BFA (5 μg/ml), and then at 37°C for 2 h (left). Alternatively, cells were incubated with 10 μM peptide and BFA, extensively washed, and then incubated at 37°C for the indicated time in the absence of peptide (right). The amount of cell-surface Kd was determined using the fluorescein-conjugated mAb SF1.1.1 (PharMingen, San Diego, CA). The concentration of peptide required for protection of one half of the Kd molecules was determined by linear extrapolation of the mean channel fluorescence values.
Mentions: 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 × 107 M−1 versus 4.2 × 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 × 104 M−1).

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