PubMed · 8566439
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A M Benham, J J Neefjes. 1995. Antigen processing by the class I pathway.. https://doi.org/10.1042/bst0230664
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Most major histocompatibility complex (MHC) class I-binding peptides are translocated by TAP heterodimers, but some enter the ER lumen by alternative pathways. To further define mechanisms of peptide handling, we developed a system for the analysis of peptide-binding components in the ER membrane and lumen using iodinated cross-linkable peptide derivatives. Here we demonstrate that at least three proteins bind peptides in the ER lumen. Peptide cross-linking to these lumenal proteins can be used as an alternative method to monitor peptide transport. TAP and one other protein bind peptides on the cytoplasmic face of the ER. The presence of multiple peptide-binding proteins necessitates caution in interpreting traditional peptide-binding and transport assays. Finally, we demonstrate sequence-specific peptide transport in TAP-deficient cells transfected with only rat TAP1.
Class I MHC heavy chains associate with many proteins in the endoplasmic reticulum, including TAP, calnexin, calreticulin, and the newly defined tapasin molecule. Recent studies have begun to resolve the nature of how these proteins interact with class I as well as the functional significance of each of these interactions. We propose here that TAP and tapasin are leading candidates to be highly specific chaperones for the class I molecule.
In an effort to optimize the formation of peptide-specific CTL target structures in the context of plasmid DNA immunization, a strategy was developed to couple the biosynthesis of a class I heavy chain with its optimal binding epitope. Specifically, a cDNA expression vector was constructed with the influenza nucleoprotein epitope NP366-74 incorporated into the signal sequence of its restriction element H-2Db. Transporter associated with Ag presentation-expressing murine cell lines P815 (H-2d) and BW5147 (H-2k) transfected with this modified heavy chain expressed normal levels of plasmid-encoded Db at the cell surface, and were lysed by NP366-74-specific CTL. These results indicate that the modified signal sequence was successfully delivered to the endoplasmic reticulum, and the epitope within it processed for T cell recognition. In contrast, T2 cells, which lack the TAP transporter, when transfected with the same vector were not lysed by NP366-74 CTL, and exhibited an Ag-processing-defective phenotype. Thus, these data, which indicate TAP-dependent presentation of an optimal CTL epitope located in a signal sequence, challenge the effectiveness of Ag processing in the endoplasmic reticulum.