Trafficking of exogenous peptides into proteasome-dependent major histocompatibility complex class I pathway following enterotoxin B subunit-mediated delivery.
The B-subunit component of Escherichia coli heat-labile enterotoxin (EtxB), which binds to cell surface GM1 ganglioside receptors, was recently shown to be a highly effective vehicle for delivery of conjugated peptides into the major histocompatibility complex (MHC) class I pathway. In this study we have investigated the pathway of epitope delivery. The peptides used contained the epitope either located at the C terminus or with a C-terminal extension. Pretreatment of cells with cholesterol-disrupting agents blocked transport of EtxB conjugates to the Golgi/endoplasmic reticulum, but did not affect EtxB-mediated MHC class I presentation. Under these conditions, EtxB conjugates entered EEA1-positive early endosomes where peptides were cleaved and translocated into the cytosol. Endosome acidification was required for epitope presentation. Purified 20 S immunoproteasomes were able to generate the epitope from peptides in vitro, but 26 S proteasomes were not. Only presentation from the C-terminal extended peptide was proteasome-dependent in cells, and this was found to be significantly slower than presentation from peptides with the epitope at the C terminus. These results implicate the proteasome in the generation of the correct C terminus of the epitope and are consistent with proteasome-independent N-terminal trimming. Epitope presentation was blocked in a TAP-deficient cell line, providing further evidence that conjugated peptides enter the cytosol as well as demonstrating a requirement for the peptide transporter. Our findings demonstrate the utility of EtxB-mediated peptide delivery for rapid and efficient loading of MHC class I epitopes in several different cell types. Conjugated peptides are released from early endosomes into the cytosol where they gain access to proteasomes and TAP in the "classical" pathway of class I presentation.
Hearn
Arron R
AR
Department of Biochemistry , School of Medical Sciences, University of Bristol, University Walk, Bristol BS8 1TD, United Kingdom.
de Haan
Lolke
L
Pemberton
Alexander J
AJ
Hirst
Timothy R
TR
Rivett
A Jennifer
AJ
D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D017951:Antigen Presentation; D002460:Cell Line; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000378:metabolism; D002784:Cholesterol / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D003600:Cytosol / Q000378:metabolism; D003713:Dendritic Cells / Q000166:cytology; D004721:Endoplasmic Reticulum / Q000378:metabolism; D011992:Endosomes / Q000378:metabolism; D004768:Enterotoxins / Q000737:chemistry*; D000939:Epitopes / Q000737:chemistry; D004926:Escherichia coli / Q000378:metabolism; D005372:Filipin / Q000494:pharmacology; D006056:Golgi Apparatus / Q000378:metabolism; D006737:Horseshoe Crabs / Q000378:metabolism; D007700:Kinetics; D008285:Major Histocompatibility Complex; D051379:Mice; D008810:Mice, Inbred C57BL; D018613:Microscopy, Confocal; D010455:Peptides / Q000737:chemistry*; D010587:Phagocytosis; D011480:Protease Inhibitors / Q000494:pharmacology; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D013997:Time Factors; D014733:Vibrio / Q000378:metabolism; D047392:beta-Cyclodextrins / Q000494:pharmacology
D000900:Anti-Bacterial Agents; D004768:Enterotoxins; D000939:Epitopes; D010455:Peptides; D011480:Protease Inhibitors; D047392:beta-Cyclodextrins; C108732:methyl-beta-cyclodextrin; C031818:enterotoxin B, staphylococcal; D005372:Filipin; D002784:Cholesterol; D046988:Proteasome Endopeptidase Complex; C061553:ATP dependent 26S protease