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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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Related in: MedlinePlus

Comparison of presentation of NP147-155 to NP50-57.  L-Kd cells infected with the rVV  indicated were tested for lysis by  TCD8+ lines specific for NP147-155  or NP50-57 at the indicated E/T.
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Figure 4: Comparison of presentation of NP147-155 to NP50-57. L-Kd cells infected with the rVV indicated were tested for lysis by TCD8+ lines specific for NP147-155 or NP50-57 at the indicated E/T.

Mentions: 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.


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)

Comparison of presentation of NP147-155 to NP50-57.  L-Kd cells infected with the rVV  indicated were tested for lysis by  TCD8+ lines specific for NP147-155  or NP50-57 at the indicated E/T.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 4: Comparison of presentation of NP147-155 to NP50-57. L-Kd cells infected with the rVV indicated were tested for lysis by TCD8+ lines specific for NP147-155 or NP50-57 at the indicated E/T.
Mentions: 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.

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