The objective of this proposal is the characterization of the structure, function and receptor biology of alpha2-macroglobulin (alpha2M), a plasma proteinase inhibitor important in the regulation of proteinases of each of the four mechanistic classes. The scope of the proposal has been narrowed from previous years where projects focused on serine proteinase inhibitors, inter-alpha-trypsin inhibitor, chemically synthesized inhibitors of coagulation factors and alpha2M. Recent studies of this and other laboratories establish a major role of alpha2M-proteinase complexes in the regulation of tumor kill, inflammation and immune function via well-established signal transduction pathways following receptor recognition of alpha2M-proteinase complexes. The focus of HL-24066 will remain on structure/function and not the signal transduction pathway. Studies during the last five years establish that the alpha2M receptor recognition site is in the carboxyl terminal. It is now proposed to study the role of this region in receptor recognition by use of expression and mutational analysis and by use of synthetic peptides. The ability of alpha2M to regulate macrophage immune function will be studied in an antigen presentation assay. The goal of these studies is to determine whether antigen uptake by macrophages is facilitated as a result of co-incorporation of antigens in alpha2M which occurs during proteinase attack on alpha2M. It is further proposed to obtain direct information on the nature of proteinases bound to alpha2M under biological conditions. These studies will be made possible by the fact that, in human cirrhosis, alpha2M-proteinase complexes circulate in plasma and can be isolated and the bound proteinase sequenced. These studies are important since all previous investigations provide only inferential evidence about the in vivo spectrum of activity of alpha2M. The final studies will further probe the evolution of the receptor recognition site of alpha2M. In conjunction with these studies, efforts will be directed towards understanding when complement precursor-like lytic activity and proteinase inhibitory activity diverged into separate pathways. Recent data from this laboratory establish that both activities reside in the same molecule, now named limac. During the course of evolution, gene duplication and divergence occurred so that a family of complement proteins which are alpha2M homologues (C3, C4 and C5) arose. Human complement proteins do not inhibit proteinases and human alpha2M does not possess a cell lytic capability.
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