Abstract
In this study we demonstrate that a disarmed version of the cytotoxin ricin can deliver exogenous CD8(+) T cell epitopes into the MHC class I-restricted pathway by a TAP-independent, signal peptidase-dependent pathway. Defined viral peptide epitopes genetically fused to the N terminus of an attenuated ricin A subunit (RTA) that was reassociated with its partner B subunit were able to reach the early secretory pathway of sensitive cells, including TAP-deficient cells. Successful processing and presentation by MHC class I proteins was not dependent on proteasome activity or on recycling of MHC class I proteins, but rather on a functional secretory pathway. Our results demonstrated a role for signal peptidase in the generation of peptide epitopes associated at the amino terminus of RTA. We showed, first, that potential signal peptide cleavage sites located toward the N terminus of RTA can be posttranslationally cleaved by signal peptidase and, second, that mutation of one of these sites led to a loss of peptide presentation. These results identify a novel MHC class I presentation pathway that exploits the ability of toxins to reach the lumen of the endoplasmic reticulum by retrograde transport, and suggest a role for endoplasmic reticulum signal peptidase in the processing and presentation of MHC class I peptides. Because TAP-negative cells can be sensitized for CTL killing following retrograde transport of toxin-linked peptides, application of these results has direct implications for the development of novel vaccination strategies.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Animals
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Antigen Presentation* / genetics
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Antigens, Viral / genetics
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Antigens, Viral / immunology
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Antigens, Viral / metabolism
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Cytotoxicity Tests, Immunologic
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Dogs
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Endoplasmic Reticulum / enzymology
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Endoplasmic Reticulum / genetics
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Endoplasmic Reticulum / immunology
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Endoplasmic Reticulum / metabolism
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Genetic Engineering / methods
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Glycoproteins / genetics
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Glycoproteins / immunology
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Glycoproteins / metabolism
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H-2 Antigens / biosynthesis
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H-2 Antigens / immunology
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H-2 Antigens / metabolism*
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Histocompatibility Antigen H-2D
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Hydrolysis
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Membrane Proteins*
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Mice
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Mice, Inbred C57BL
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Peptide Fragments / genetics
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Peptide Fragments / immunology
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Peptide Fragments / isolation & purification
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Peptide Fragments / metabolism*
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Protein Processing, Post-Translational / genetics
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Protein Processing, Post-Translational / immunology*
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Protein Transport / genetics
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Protein Transport / immunology
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Rabbits
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Recombinant Fusion Proteins / chemical synthesis
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Recombinant Fusion Proteins / immunology
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Recombinant Fusion Proteins / metabolism
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Ricin / genetics
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Ricin / immunology
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Ricin / metabolism*
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Serine Endopeptidases / metabolism*
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Tumor Cells, Cultured
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Viral Core Proteins / genetics
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Viral Core Proteins / immunology
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Viral Core Proteins / metabolism
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Viral Proteins / genetics
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Viral Proteins / immunology
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Viral Proteins / metabolism
Substances
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Antigens, Viral
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Glycoproteins
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H-2 Antigens
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Histocompatibility Antigen H-2D
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Membrane Proteins
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Peptide Fragments
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Recombinant Fusion Proteins
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Viral Core Proteins
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Viral Proteins
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glycoprotein peptide 33-41, Lymphocytic choriomeningitis virus
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nucleoprotein (366-374), influenza virus
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Ricin
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Serine Endopeptidases
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type I signal peptidase