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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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Characterization of  SNP. (A) Aliquots of the target  cells used in Fig. 3 A were removed 90 min after infection  and radiolabeled with [35S]methionine. Total cell extracts were  analyzed by SDS-PAGE. Radioactivity in the fixed and dried  gels was located using a Phosphorimager that was used to  quantitate the radioactivity in the  NP and SNP band, which are  clearly evident. The total  amounts of counts per lane were  normalized to allow for differences in sample preparation, and  the corrected amount of radioactivity present in the same region  of an extract prepared from cells  infected with a control VV was  subtracted from each value. The  adjusted amounts of various  forms of NP synthesized relative  to NP (= 1.00) are: NPN, 1.65;  NPNRA, 2.16; SNP, 1.24;  SNPN, 1.72; SNPN high, 2.23;  and SNPNRA, 1.82. (B) NP  present in detergent lysates from  cells infected with rVV expressing various forms of NP was collected using protein A agarose  preloaded with the NP-specific  mAb H16-L10, digested with  endo H and analyzed by SDS-PAGE.
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Figure 2: Characterization of SNP. (A) Aliquots of the target cells used in Fig. 3 A were removed 90 min after infection and radiolabeled with [35S]methionine. Total cell extracts were analyzed by SDS-PAGE. Radioactivity in the fixed and dried gels was located using a Phosphorimager that was used to quantitate the radioactivity in the NP and SNP band, which are clearly evident. The total amounts of counts per lane were normalized to allow for differences in sample preparation, and the corrected amount of radioactivity present in the same region of an extract prepared from cells infected with a control VV was subtracted from each value. The adjusted amounts of various forms of NP synthesized relative to NP (= 1.00) are: NPN, 1.65; NPNRA, 2.16; SNP, 1.24; SNPN, 1.72; SNPN high, 2.23; and SNPNRA, 1.82. (B) NP present in detergent lysates from cells infected with rVV expressing various forms of NP was collected using protein A agarose preloaded with the NP-specific mAb H16-L10, digested with endo H and analyzed by SDS-PAGE.

Mentions: 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).


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)

Characterization of  SNP. (A) Aliquots of the target  cells used in Fig. 3 A were removed 90 min after infection  and radiolabeled with [35S]methionine. Total cell extracts were  analyzed by SDS-PAGE. Radioactivity in the fixed and dried  gels was located using a Phosphorimager that was used to  quantitate the radioactivity in the  NP and SNP band, which are  clearly evident. The total  amounts of counts per lane were  normalized to allow for differences in sample preparation, and  the corrected amount of radioactivity present in the same region  of an extract prepared from cells  infected with a control VV was  subtracted from each value. The  adjusted amounts of various  forms of NP synthesized relative  to NP (= 1.00) are: NPN, 1.65;  NPNRA, 2.16; SNP, 1.24;  SNPN, 1.72; SNPN high, 2.23;  and SNPNRA, 1.82. (B) NP  present in detergent lysates from  cells infected with rVV expressing various forms of NP was collected using protein A agarose  preloaded with the NP-specific  mAb H16-L10, digested with  endo H and analyzed by SDS-PAGE.
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Related In: Results  -  Collection

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Figure 2: Characterization of SNP. (A) Aliquots of the target cells used in Fig. 3 A were removed 90 min after infection and radiolabeled with [35S]methionine. Total cell extracts were analyzed by SDS-PAGE. Radioactivity in the fixed and dried gels was located using a Phosphorimager that was used to quantitate the radioactivity in the NP and SNP band, which are clearly evident. The total amounts of counts per lane were normalized to allow for differences in sample preparation, and the corrected amount of radioactivity present in the same region of an extract prepared from cells infected with a control VV was subtracted from each value. The adjusted amounts of various forms of NP synthesized relative to NP (= 1.00) are: NPN, 1.65; NPNRA, 2.16; SNP, 1.24; SNPN, 1.72; SNPN high, 2.23; and SNPNRA, 1.82. (B) NP present in detergent lysates from cells infected with rVV expressing various forms of NP was collected using protein A agarose preloaded with the NP-specific mAb H16-L10, digested with endo H and analyzed by SDS-PAGE.
Mentions: 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).

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