This proposal is to continue the examination of the processing and presentation of protein antigens. The main goal is to obtain information on the biochemistry and cellular basis of protein processing by antigen presenting cells (APC) that results in the surface expression of a peptide associated with class II-MHC molecules (Ia). We focus here on the identification of subcellular pathways of antigen transport and on the biochemical analysis of protein antigens; in the identification of organelles where proteins are fragmented and where the fragments associated with Ia; and in the analysis of conditions that may modulate the primary interaction of Ia with peptides. We combine herewith techniques, many already developed, of cellular fractionation, peptide and protein biochemistry and cellular immunology. The goal of Project 1 is to identify, isolate and characterize intracellular organelles that bear Ia molecules. It includes ultrastructural analysis and biochemical attempts to isolate Ia-bearing vesicles. It also contains biochemical attempts to reproduce Ia-peptide interactions in cell-free systems using the contents of, or the conditions found in, endosomes. It has a section evaluating the role of Invariant chain. The goal of Project 2 is to identify biochemically the products of catabolism of the protein hen-egg white lysozyme relevant in antigen presentation. Included are several projects examining the intracellular site of catabolism of antigen and of coupling of peptide to Ia. A key goal is to identify the chemistry of natural peptides of lysozyme that associate with Ia molecules. The goal of Project 3 is to examine whether there are differences in antigen processing among APC that depend on cell types and/or their state of activation. It also includes studies examining how polysaccharides affect antigen processing. The information on the biochemical and cellular basis of antigen processing and presentation will allow us to understand many of the events, and regulation, of immune induction and some of the basis for self/non-self identification of proteins.
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