title: Exogenous peptides delivered by ricin require processing by signal peptidase for transporter associated with antigen processing-independent MHC class I-restricted presentation.
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.
authors.affiliation: Department of Biological Sciences, University of Warwick, Coventry, United Kingdom.
authors.forename: Daniel C
authors.initials: DC
authors.lastname: Smith
authors.forename: Awen
authors.initials: A
authors.lastname: Gallimore
authors.forename: Emma
authors.initials: E
authors.lastname: Jones
authors.forename: Brenda
authors.initials: B
authors.lastname: Roberts
authors.forename: J Michael
authors.initials: JM
authors.lastname: Lord
authors.forename: Emma
authors.initials: E
authors.lastname: Deeks
authors.forename: Vincenzo
authors.initials: V
authors.lastname: Cerundolo
authors.forename: Lynne M
authors.initials: LM
authors.lastname: Roberts
mesh_terms: D000818:Animals; D017951:Antigen Presentation / Q000235:genetics; D000956:Antigens, Viral / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D003601:Cytotoxicity Tests, Immunologic; D004285:Dogs; D004721:Endoplasmic Reticulum / Q000201:enzymology / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D005818:Genetic Engineering / Q000379:methods; D006023:Glycoproteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D006183:H-2 Antigens / Q000096:biosynthesis / Q000276:immunology / Q000378:metabolism*; D064532:Histocompatibility Antigen H-2D; D006868:Hydrolysis; D008565:Membrane Proteins; D051379:Mice; D008810:Mice, Inbred C57BL; D010446:Peptide Fragments / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D011499:Protein Processing, Post-Translational / Q000235:genetics / Q000276:immunology*; D021381:Protein Transport / Q000235:genetics / Q000276:immunology; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000138:chemical synthesis / Q000276:immunology / Q000378:metabolism; D012276:Ricin / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D012697:Serine Endopeptidases / Q000378:metabolism*; D014407:Tumor Cells, Cultured; D014758:Viral Core Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D014764:Viral Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism
chemical_list: D000956:Antigens, Viral; D006023:Glycoproteins; D006183:H-2 Antigens; D064532:Histocompatibility Antigen H-2D; D008565:Membrane Proteins; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D014758:Viral Core Proteins; D014764:Viral Proteins; C094244:glycoprotein peptide 33-41, Lymphocytic choriomeningitis virus; C102366:nucleoprotein (366-374), influenza virus; D012276:Ricin; D012697:Serine Endopeptidases; C026515:type I signal peptidase