Schistosomiasis is a major worldwide health problem, with an estimated 250 million people in 76 countries suffering from chronic infection. Human infection is initiated through skin penetration by schistosomal larvae, which has been shown to be mediated by a secreted serine protease, the cercarial elastase. This proposal focuses on understanding the cercarial elastase as a pivotal virulence factor of invading schistosomes. The long term objective of this proposal is to determine the molecular basis of the proteolytic capacity of the cercarial elastase by identifying the unique structural elements that confer its substrate specificity. This will facilitate the rational design of specific inhibitors, and will be relevant to the pathogenesis of other tissue-invasive parasitic diseases, as well as to other pathologic processes involving proteolytic tissue destruction such as inflammation and tumor metastasis. In studying the cercarial elastase as a model serine protease, we will enhance the understanding of the structure-function basis of proteolytic specificity for this important class of enzymes.
Our specific aims are to express recombinant cercarial elastase in E. coli using expression strategies successful for other serine proteases. The recombinant enzyme will be used to precisely map the substrate specificities to synthetic peptides, guiding the design of peptide-based inhibitors. The structural determinants of the cercarial elastase that confer its broad proteolytic specificity will be characterized, by using a homology-based structural model in conjunction with site directed mutagenesis to generate mutant enzymes with catalytic activity but altered substrate specificity. These mutant elastases will be used to examine the basis for the proteolysis of macromolecular substrates in the skin such as elastin. The purification and X-ray crystallographic structure determination of the expressed cercarial elastase will be pursued in parallel, allowing confirmation and enhancement of the theoretical model.
| Sun, E; Cohen, F E (1993) Computer-assisted drug discovery--a review. Gene 137:127-32 |
