Elastic tissue degradation is a major component in the pathogenesis of emphysema and atherosclerosis. We have previously isolated and characterized pancreatic elastase 2, the major elastolytic enzyme in man. During the funding period we have obtained evidence that pancreatic proelastase 2 enters the bloodstream in significant amounts. We hypothesize that tissue sequestration and subsequent activation of circulating proelastase 2 may be an important factor in destruction of tissue elastin in disease processes. Elastases are unique members of a large family of closely related serine proteases, due to their capacity to degrade elastin. The detailed structural properties which confer this unique ability to degrade elastin is unclear for all known elastases, and is particularly ill-defined for human pancreatic elastase 2. The advent of recombinant DNA and site specific mutagenesis techniques make possible, for the first time, the detailed elucidation of structural parameters controlling elastolysis. The specific goal of this project is to determine the individual amino acids in the primary structure of human elastase 2 which confer this property. In order to accomplish this goal, we propose to clone elastase 2 from an existing human pancreatic cDNA library. The nucleotides corresponding to particular amino acids in the elastase 2 primary structure will be changed using site-specific mutagenesis. The original cloned elastase 2 as well as the modified enzymes will be expressed in an SV40/COS monkey kidney cell system, and assayed for elastolytic activity. It is anticipated that this approach will reveal the primary structure elements necessary for elastolysis, and will facilitate the design of specific inhibitors for human pancreatic elastase 2. In addition, the structural data obtained may be applicable to an increased understanding of the structure/function relationship of tissue elastases.
