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Aquatic larvae (cercariae) of the trematode parasite Schistosoma mansoni rapidly penetrate human skin by degrading host proteins including elastin. Two serine proteases, one chymotrypsin-like and the second trypsin-like, have been proposed to be involved. To evaluate the relative roles of these two proteases in larval invasion, both were purified, identified by sequence, and then biochemically characterized. The trypsin-like activity was resolved into two distinct serine proteases 76% similar in predicted amino acid sequence. Southern blot analysis, genomic polymerase chain reaction, and immunolocalization demonstrated that the trypsin-like proteases are in fact not from the schistosome, but are released with larvae from the snail host Biomphalaria glabrata. Invasion inhibition assays using selective inhibitors confirmed that the chymotrypsin-like protease 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.Infection of a human host by the trematode parasite Schistosoma mansoni begins with invasion of intact skin by an aquatic larva, the cercaria (1). Exiting the freshwater snail Biomphalaria glabrata, cercariae locate a human host by thermal (2) and chemical (3) signals and rapidly penetrate the skin, entering the vascular system in the dermis (4). S. mansoni cercariae are ϳ150 m long and 70 m wide and require lysis of skin tissues to migrate into blood vessels. Host macromolecules representing barriers to cercarial invasion are known to be cleaved by proteolytic activities present in cercarial secretions. These include elastin (5); chondromucoprotein (6); keratin (7); fibronectin, laminin, and collagens IV and VIII (8). Two distinct serine proteases have been reported in extracts of cercariae or in secretions from cercariae induced upon contact with skin lipids. One is a "chymotrypsin-like" protease with a preference for large hydrophobic side chains at P1 (9). The second is a "trypsin-like" protease with a preference for positively charged side chains at P1 (10). The class of proteases responsible for host protein degradation has been demonstrated by several independent studies to be serine proteases (6), but the relative contributions to invasion of the trypsin-like or chymotrypsin-like proteases are not known.To analyze the relative contributions of each of these proteases to the degradation of host proteins, cercarial secretions were fractionated, and the two proteases purified. The trypsinlike activity, which had not been previously purified or sequenced, was purified, and a cDNA was cloned by reverse transcription-PCR 1 based on amino-terminal amino acid sequence. Specific inhibitors were identified to evaluate the role of each protease in skin invasion. Proteases were localized by immunohistochemistry, and specific sites of cleavage in elastin, the most protease-resistant target in host skin, were analyzed.

Water borne cercaria(ae) of the trematode genus Schistosoma rapidly penetrate host skin. A single serine protease activity, cercarial elastase, is deposited in advance of the invading parasite by holocytosis of vesicles from ten large acetabular gland cells. Cercarial elastase activity is a composite of multiple isoforms. Genes coding for the isoforms 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% of the activity and protein released. The remaining genes produce little protein or are silent. Positional scanning synthetic combinatorial substrate libraries demonstrate that the two major isoforms have similar substrate specificities and are, therefore, isoenzymes. The closely related Schistosoma hematobium and the distantly related Schistosomatium douthitti also contain multiple orthologous cercarial elastase genes suggesting that gene duplication may have occurred after speciation in Schistosoma evolution and that this duplication has been conserved.Cercaria(e), the aquatic infective larval stage of schistosomes, are highly adapted to rapidly penetrate the skin of the host upon contact. Enzymatic hydrolysis of host proteins is required for successful entry into the host vascular system (1). Two gland systems, the preacetabular and postacetabular glands, release proteases and comprise the majority of the volume of the cercarial head. Each gland cell releases proteases at the leading edge of the invading parasite through long, microtubule-lined cell processes or "ducts" that exit at the anterior head (2). The postacetabular glands are also responsible for depositing mucin, providing an adhesive surface on the skin for the parasite to initially attach. Considering the diverse set of macromolecular barriers the cercariae must breach during invasion, we previously investigated the possibility that multiple enzyme activities were required. However, only a single protease activity, cercarial elastase, was found to be present in acetabular gland secretions and required for invasion (3).Cercarial elastase is a trypsin family serine protease named because of its ability to cleave insoluble elastin, a major component of the dermis of skin (4, 5). Its P1 substrate specificity (1) is for large hydrophobic side chains, but in contrast to chymotrypsin (3) cercarial elastase is more active against macromolecular substrates than synthetic tetrapeptides.We examined the complement of genes coding for cercarial elastase in Schistosoma mansoni and found a family of isoforms that can be divided into two classes by amino acid and promoter sequence homology. This family of genes is also conserved in another schistosome species Schistosoma hematobium and Schistosomatium douthitti. The two most highly expressed S. mansoni isoforms comprise Ͼ90% of the released activity and are virtually identical in biochemical properties.

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