Identification and characterization of thiol proteases released in vitro by Fasciola hepatica

Fasciola hepatica excretory/secretory (E/S) products were obtained by culturing liver flukes invitro for 16 hours. When these E/S products were analyzed by gelatin substrate sodiumdodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) two groups of proteases wererevealed. Group 2 proteases were active in the pH range 4.5-8.0, while Group 1 proteaseswere most active in the pH range 3.0-4.5. Activity of all proteases was enhanced by thereducing agents cysteine and DTT. Based on these studies and on inhibitor studies bothgroups of proteases were identified as thiol proteases. Also consistent with thiol proteases all o f the enzymes bound to an organomercurial column. Antiserum against these thiol proteases eluted from this column was prepared. Using this antiserum in immunolocalization studies we showed that these enzymes are secreted by the gut epithelial cells. The antiserum was also used to screen an adult fluke cDNA expression library; however, no recombinant clones were identified. Rabbit IgG was incubated with the E/S products or papain. Analysis by SDSPAGE revealed that both preparations resulted in the cleavage of IgG heavy chain into two fragments o f 28 and 22 kDa. HPLC analysis o f the E/S products yielded three major peaks. The IgG cleaving enzyme was found to be associated with peak m of approximately 15 kDa. Using the substrate Z-Phe-Arg-AMC we showed that this peak also contained cathepsin B activity. Consistent with cathepsin B enzymes the enzyme in peak m was found to be a thiol protease with optimal activity at pH 4.5. Gelatin substrate PAGE analysis showed that peak IQ correlated with the acidic Group 1 proteases. DPC, which inhibits enzymes containing histidine in their active site, totally inactivated the enzyme in peak IQ and inhibited the cleavage o f IgG by this enzyme. This result is consistent with this enzyme being categorized as a cathepsin B since these enzymes have histidine residues involved in catalytic activity