PURIFICATION AND CHARACTERIZATION OF A 2ND CATHEPSIN-L PROTEINASE SECRETED BY THE PARASITIC TREMATODE FASCIOLA-HEPATICA

A 29.5-kDa cysteine proteinase was purified from medium in which matur e Fasciola hepatica parasites were maintained. The N-terminal sequence (14 residues) of the purified protein is similar to known cathepsin L proteinases, including a 27-kDa cathepsin L proteinase, also secreted by this parasite, which had been isolated previously in our laborator y [Smith, A. M., Dowd, A, J., Me Gonigle, S., Keegan, P. S., Brennan, G., Trudgett, A. and Dalton, J. P. (1993) Mel. Biochem. Parasitol. 62, 1-8]. The N-terminal sequences of the 29.5-kDa and 27-kDa cathepsin L proteinases differ only in residue number seven (arginine and proline , respectively). Immunoblot studies, using antiserum that reacts with both cathepsin L proteinases, rule out the possibility of both enzymes arising from a higher molecular sized parent molecule. The reaction k inetics of the two F. hepatica cathepsin L proteinases on a variety of peptide substrates revealed that the two enzymes differ in their subs trate specificity. Five peptide substrates that are cleaved with high affinity by the 29.5-kDa cathepsin L isolated in this study are not cl eaved by the previously purified 27-kDa cathepsin L. The protein-modif ying reagent, tetranitromethane, affected the 29.5-kDa cathepsin L pro teinase only, causing inactivation of the enzyme and changing its migr ation in polyacrylamide gel electrophoresis. Our studies suggest that the two F. hepatica cysteine proteinases represent two distinct subcla sses within the cathepsin L class.