Alpha1-Proteinase inhibitor (A1Pi) deficiency is a common human genetic disease associated with increased risk of developing pulmonary emphysema. Emphysema in individuals suffering from this disease appears to be a result of low serum levels of A1Pi and as a consequence a decreased ability to inhibit elastase released from activated neutrophils. This leads to excessive degradation of the elastic lung tissue and eventually emphysema. In almost all cases the low serum levels of A1Pi are due to inefficient secretion of mutant forms of A1Pi from hepatocytes, their major site of synthesis. The defect in secretion is a direct consequence of the mutation in the A1Pi gene and appears to be due to structural changes which render the A1Pi variant """"""""secretion incompetent"""""""". Establishing the means by which these mutations affect secretion is the major objective of this proposal. The approaches toward achieving this goal include production of A1Pi variants by site directed mutagenics; establishing the efficiencies of secretion and the intracellular stabilities of these variants in transiently transfected and stably transformed cell lines; ultrastructural comparisons of cells stably expressing normal and variant forms of A1Pi in an effort to detect differences in their sites of accumulation within organelles, especially within the ER; production of adequate amounts of these variants to allow physical studies on their structures; and to identify components of the exocytic pathway whose interactions with A1Pi may be altered by the mutations. Variants have been, or will be, produced by oligonucleotide directed mutagenesis of A1Pi cDNA. The fates of the gene products will be established by pulse chase experiments and subsequent analyses by appropriate means. Cell lines for the ultrastructural studies have been constructed and additional lines will be produced as needed. Quantities of the A1Pi variants needed for physical studies will be produced in cultures of sf9 cells infected with appropriate recombinant baculoviruses. Changes in the conformations of variants will be monitored by spectroscopic methods. Intracellular components that interact with A1Pi will be studied by co-immunoprecipitation, with and without chemical cross-linking. It is hoped that these studies will lead to an understanding of the molecular basis for the defective secretion of A1Pi in individuals suffering from the deficiency disease.
