This is an application for continuing support of our studies on the function of a mouse major histocompatibility complex (MHC) class I-b molecule, H-2M3. We previously established that this molecule has a peptide binding specificity that is unique in its requirement for N- formylated peptides. This finding led us during the previous period of support to work that has contributed importantly to establishing H-2M3 as the first class I-b molecule for which a unique role in antigen presentation has been defined. The studies proposed in this renewal application are directed to evaluating critically the hypothesis that H- 2M3 represents a specialization of MHC function for defenses to infection by prokaryotic organisms. The approach will combine molecular and genetic strategies with analysis at the level of whole cells and intact organisms.
Four specific aims are proposed. First, we will further define the specificity of the H-2M3 peptide binding cleft. Experiments are proposed that should lead to a clearer understanding of the N-formyl peptide-MHC interaction, both through detailed analysis of specificity of MHC pocket A mutants and study of peptides eluted from H-2M3 affinity columns. Second, we will investigate the processing and transport of antigens synthesized by or derived from prokaryotic organisms for presentation by H-2M3. From these experiments we hope to learn how N-formylated peptides progress from the bacteria to the H-2M3-binding compartment within the endoplasmic reticulum. Third, we will explore the postulated role of H-2M3 in prokaryotic host defenses. Our approaches will include the use of transgenic mice expressing H-2M3 as their predominant class I MHC molecule, mice expressing an H-2M3-restricted T cell receptor and bacteria engineered to express an N-formylated peptide for which the transgenic TCR has specificity. Finally, experiments are proposed to investigate the possibility of non-Murid analogues for H-2M3, including humans. Gene-based approaches will be employed in an effort to identify possible orthologous sequences to H-2M3 among class 1-b genes of non-Murid species. An independent protein-based search for MHC molecules that bind formylated peptides will be used to screen for non-orthologous functional homologs. These multi-faceted studies should, we believe, provide novel insights into the role of the MHC in host defenses and may eventually prove applicable to strategies for vaccine design in humans.
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