.) The long term goal of this project is to define structural features of alpha-1 proteinase inhibitor (A1Pi) required to allow its secretion at levels adequate for protection against damage to the lower respiratory tract due to the action of elastase.
The specific aims are to determine the fates and properties of A1Pi variants produced by recombinant DNA techniques which are analogous to naturally occurring variants known to cause human disease or that have changes in regions suspected to play a role in the fate of the protein after it enters the lumen of the endoplasmic reticulum. To accomplish these goals, variants that have already been produced by oligonucleotide-directed mutagenesis, or that will be produced during the course of this project, will be characterized with respect to their ability to inhibit serine proteases, their rates of secretion and/or degradation, and their physical properties. Biological activity will be measured by standard assays for inhibition of serine protease activity and by characterization of the covalent adducts formed between the different forms of A1Pi and serine proteases using SDS-PAGE. Rates of secretion will be measured in pulse chase experiments and/or ELISA. To produce adequate amounts of the variant forms of A1Pi for physical characterization, the corresponding cDNA's will be inserted into vectors for expression in Escherichia coli and into the baculoviral system for expression in insect cells. The variant proteins produced in these systems will be isolated by standard procedures and characterized by measuring changes in their spectroscopic properties during unfolding and refolding resulting from the addition, and subsequent removal, of denaturing agents. In addition, the interactions of these proteins with conformation specific monoclonal antibodies will be examined and their susceptibility to various nonserine proteases will be determined. The results of these experiments will be interpreted within the context of different models which may explain the defective secretion of some of the variants of A1Pi that are associated with pulmonary and liver disease. The results may also be applicable to the general area of protein secretion and targeting.
