A majority of patients with Wegener's Granulomatosis (WG) have circulating autoantibodies directed against an intracellular neutrophil/monocyte antigen of 29 kD (p29, PR3, Wegener's autoantigen) present in the azurophilic granules. During the previous funding period we elucidated the serine proteinase nature of p29 and established its diagnostic value in patients with systemic vasculitis syndromes. We have also shown in a small sample of patients with active WG that a majority has autoantibodies that inhibit the proteolytic activity of p29 in vitro. We also found that following neutrophil activation, a significant component of p29 can associate with the cell surface membrane. This association does not require a catalytically-active enzyme, and therefore appears distinct from a SEC (serpin-enzyme complex) receptor-mediated uptake. We have modeled the 3-D structure of human p29 based on the crystal structure of the homologous human neutrophil elastase. This analysis revealed distinct features of p29 with regard to the surface hydropathy and catalytic pocket that may be relevant to its association with the plasma membrane, its substrate specificity and its reduced susceptibility to elastase inhibitors. p29 has also been shown to have potent proteolytic, proliferative and anti-microbial activities in vitro. The physiologic role(s) of p29, the mechanisms that regulate its expression and activity and the role autoantibodies to this enzyme play in the pathogenesis of granulomatous inflammation are unknown. The main objectives of the current proposal are: 1) To establish the relationship between the autoantibody inhibitory profile and disease activity in WG. 2) To characterize the nature of the membrane association of p29, its effect on catalysis, the nature of the surface binding moieties, and the region of p29 involved in this interaction. 3) Examine the fate of the internalized p29 when presented alone, in complex with a serpin or an autoantibody. Immunological, biochemical, cell and molecular biology techniques will be employed. The ultimate goal of these studies is to elucidate the structure and physiologic function of p29, and clarify the potential role of anti-p29 autoantibodies in granulomatous inflammation. These studies will also facilitate the development of effective and selective inhibitors of this enzyme that could prevent its tissue damaging effects in other diseases such as cystic fibrosis and emphysema.
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