| Aquatic larvae (cercariae) of the trematode parasite, Schistosoma mansoni, rapidly penetrate human skin by disrupting the epidermal cell layer and degrading host basement membrane and extracellular matrix proteins. Previous work from several laboratories identified two serine proteases potentially involved in cercarial invasion, one "chymotrypsin-like" (cercarial elastase) and the second "trypsin-like". To evaluate the relative roles of these two proteases in larval invasion both were purified, and biochemically characterized. Invasion inhibition assays using selective inhibitors confirmed that cercarial elastase is the enzyme involved in skin penetration. Its ability to degrade skin elastin was confirmed and the three sites of cleavage within elastin help define a new family of elastases.;Genes coding for cercarial elastase isoforms were identified and sequenced. They can be divided into two classes by amino acid and promoter sequence homology. Two of the five genes identified in Schistosoma mansoni account for over 90 percent of the enzyme released. Positional scanning synthetic combinatorial libraries demonstrate that the two major isoforms are isoenzymes.;Previous work suggested that cercarial elastase may have extended binding sites on both the amino terminal and carboxy terminal side of peptide bond cleavage. To test this hypothesis, positional scanning synthetic combinatorial libraries and a substrate phage library were used to map selectivity from P4 to P2'. A database of human proteins was then screened for potential substrates that contained the optimal substrate sequence. A number of epidermal extracellular matrix, and basement membrane proteins found in may be the motor protein that transports Spa2p and its binding partners to sites of growth and that Spa2p may also serve as a docking protein for Myo2p at the sites of growth. Second, a genetic approach was used to gain insight into the functional relationships of these polarity proteins. Although these proteins appear to act together in their polarity function, our investigations revealed that distinctions could be made among them with respect to the behavior of the mutants. spa2, pea2, bud6, and bni1 mutants exhibited some differences in growth rate, shmoo morphology, mating efficiency and localization dependence. |
