ABSTRACT
Many melanoma epitopes are presented to cytotoxic T-lymphocytes (CTLs) by major histocompatibility complex (MHC) class I molecules, and it is reasonable to expect that the epitopes would be good substrates for the transporter associated with antigen processing (TAP), as TAP plays a major role in the transport of peptides into the endoplasmic reticulum (ER) for binding to MHC class I molecules. However, we have previously shown that several melanoma-associated epitopes, such as those derived from tyrosinase, gp100, MAGE-1 and MAGE-2 antigens, are in fact poor substrates for TAP. During the process of determining why these epitopes were capable of eliciting a strong CTL response, yet were poor substrates for TAP, it was observed that the epitopes possessed amino acids at their N-terminus that were deleterious for TAP binding as described for the peptide-binding motif for human TAP. We therefore postulated that the epitopes were transported by TAP as longer precursor molecules, and then trimmed in the ER to the appropriate size for presentation to T-cells. In an effort to test this hypothesis we synthesized a set of peptides, derived from the tyrosinase (YMNGTMSQV) and MAGE-1 (EADPTGHSY) epitopes, which possess N-terminal extensions of up to four amino acids. We show here that the longer peptides are indeed transported into the ER at a significantly higher level than the original epitopes. The data indicate that the longer melanoma-associated peptides are the preferred substrates for TAP, and further support the notion that peptides can be trimmed at the N-terminus in the ER during antigen processing.
Subject(s)
ATP-Binding Cassette Transporters/metabolism , Antigen Presentation , Antigens, Neoplasm/metabolism , Endoplasmic Reticulum/metabolism , Epitopes/metabolism , Peptide Fragments/metabolism , ATP Binding Cassette Transporter, Subfamily B, Member 2 , ATP Binding Cassette Transporter, Subfamily B, Member 3 , Antigens, Neoplasm/chemistry , Antigens, Neoplasm/immunology , Biological Transport , Burkitt Lymphoma/pathology , Epitopes/chemistry , Humans , Melanoma-Specific Antigens , Microsomes/metabolism , Molecular Weight , Monophenol Monooxygenase/chemistry , Monophenol Monooxygenase/immunology , Monophenol Monooxygenase/metabolism , Neoplasm Proteins/chemistry , Neoplasm Proteins/immunology , Neoplasm Proteins/metabolism , Peptide Fragments/chemistry , Peptide Fragments/immunology , Substrate Specificity , Tumor Cells, CulturedSubject(s)
ATP-Binding Cassette Transporters/metabolism , Peptides/metabolism , ATP Binding Cassette Transporter, Subfamily B, Member 2 , ATP Binding Cassette Transporter, Subfamily B, Member 3 , ATP-Binding Cassette Transporters/analysis , ATP-Binding Cassette Transporters/chemistry , Amino Acid Sequence , Bacterial Proteins , Binding, Competitive , Biological Transport , Cell Line , Cell Membrane Permeability , Cells, Cultured , Chromatography, Affinity , Glycosylation , Humans , Kinetics , Major Histocompatibility Complex , Melanoma , Molecular Sequence Data , Peptides/chemistry , Streptolysins , Tumor Cells, CulturedABSTRACT
To gain insight into how tumor antigens are generated and presented, a panel of peptides corresponding to melanoma-specific T cell epitopes were tested for their transport capacity by the transporter associated with antigen processing (TAP). The melanoma epitopes exhibited differential capacities to be transported by TAP in streptolysin O-permeabilized cells, as well as differential competition for peptide binding to TAP. The data indicate that some melanoma-specific epitopes are good substrates for TAP, while others are poor substrates for TAP. One of the epitopes, derived from tyrosinase, was transported into the endoplasmic reticulum (ER), in spite of being a poor competitor for reporter peptide transport and for peptide binding. These results suggest that the melanoma antigens follow distinct pathways for presentation, along the MHC class I pathway.
