PEPTIDE BINDING AND EXPRESSION OF MHC CLASS I MOLECULES

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The current consensus view of antigen presentation by major histocompatibility complex (MHC) class I molecules is that these molecules bind peptides derived from foreign or self-proteins synthesized within the presenting cell. The peptides are then presented in association with MHC class I molecules to antigen-specific, MHC class I molecule-restricted T cell receptor on CD8+ cytotoxic T lymphocytes (CTL); this interaction ultimately should result in cytolysis of the presenting or target cell by CTL. Crystallographic analyses of MHC class I molecules revealed a groove into which peptides are proposed to be bound and presented; furthermore, these studies suggested potential residues in the groove of MHC class I molecules that could interact with antigenic peptides. Studies performed by Townsend et al. have implicated a role for antigenic peptide in the folding and assembly of class I molecules; furthermore, these studies suggested that this process led to egress from the ER to the cell surface. However, recent studies by Ploegh and coworkers have generated a controversy concerning the mechanism(s) (and its physiological relevance) by which that process can occur, since they were able to show that class I molecules are capable of peptide-induced assembly at the cell surface. We have previously demonstrated that the murine class I molecule, Ld, is transported to the cell surface more slowly, associates with beta2-m less strongly and displays lower surface expression than most other MHC class I molecules; Hansen and colleagues showed that the surface expression of Ld could be increased upon incubation of cells with Ld-restricted antigenic peptides. Recently, we characterized a mAb that recognizes an altered form of Ldalt that may represent an unassembled precursor of Ld. Thus, Ld represents an ideal model system to study the role of antigen presentation in surface expression of MHC class I molecules. The first objective of the proposal is to determine which amino acid residues of Ld are responsible for its expression phenotype by analyzing the expression of Ld-like molecules, and by creating and studying the expression of chimeric and mutant Ld molecules. Second, to resolve the above controversy, we propose to assess the role of aforementioned Ldalt and beta2-m in the assembly process, and to examine the kinetics if the in vivo peptide-induction process. Third, we propose to analyze the interaction of Ld, Ld-like, and mutant Ld molecules with antigenic peptides and substituted peptides in several different assays including an in vitro peptide binding assay. These analyses should determine which Ld residues are involved in peptide binding and should lead to be a better understanding of the "topography" of the peptide-class I molecule interaction. Collectively, completion of these studies should help elucidate the role that antigenic peptides play in surface expression of and subsequent antigen presentation by class I molecules and as well as the residues on class I molecules involved in these processes.