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.
Introduction of a Glycosylation Site into a Secreted Protein Provides Evidence for an Alternative Antigen Processing Pathway: Transport of Precursors of Major Histocompatability Complex Class I–Restricted Peptides from the Endoplasmic Reticulum to the Cytosol
Address correspondence to J.R. Bennink or J.W. Yewdell, Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, Bethesda, MD 20892-0440. Phone: 301-402-4602; FAX: 301-402-7362; E-mail: [email protected], [email protected]. G. Russ is a visiting scientist from the Slovak Academy of Sciences, 82446 Bratislava, Slovakia.
Beth Buschling provided excellent technical assistance. We are grateful to Dr. B. Rouse (University of Tennessee, Nashville, TN) for providing VV-ICP47 and Dr. F. Momburg (German Cancer Research Center, Heidelberg, Germany) for providing Kd-transfected T2 cells.
Abbreviations used in this paper: BFA, brefeldin A; endo H, endo-β-N-acetylglucosaminidase; ER, endoplasmic reticulum; NP, nucleoprotein; PNGase, peptide N glycanase; rVV, recombinant VV; TAP, transporter associated with antigen presentation; VV, vaccinia virus.
I. Bačík, H.L.Snyder, and L.C. Antón contributed equally to this work, and are listed in random order.
Igor Bačík, Heidi Link Snyder, Luis C. Antón, Gustav Russ, Weisan Chen, Jack R. Bennink, Laszlo Urge, Laszlo Otvos, Boguslawa Dudkowska, Laurence Eisenlohr, Jonathan W. Yewdell; Introduction of a Glycosylation Site into a Secreted Protein Provides Evidence for an Alternative Antigen Processing Pathway: Transport of Precursors of Major Histocompatability Complex Class I–Restricted Peptides from the Endoplasmic Reticulum to the Cytosol . J Exp Med 18 August 1997; 186 (4): 479–487. doi: https://doi.org/10.1084/jem.186.4.479
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