FASCIOLA-HEPATICA - CHARACTERIZATION AND CLONING OF THE MAJOR CATHEPSIN-B PROTEASE SECRETED BY NEWLY EXCYSTED JUVENILE LIVER FLUKE

Proteolytic activity present in the excreted/secreted (ES) material of newly excysted juvenile (NEJ) Fasciola hepatica was biochemically ana lyzed. By gelatin substrate SDS-PAGE, only one region of activity was observed in the NEJ ES material at a molecular mass of 29 kDa. Both th e secreted cathepsin L from adult fluke and the 29-kDa proteolytic act ivity of NEJ ES show a common pH optimum of 7.5, a cysteine protease i nhibition profile, and preference for the N-benzyloxycarbonyl (Z)-Phe- Arg-NHMec fluorogenic substrate over Z-Arg-Arg-NHMec and Z-Arg-NHMec. In vitro analysis revealed that the NEJ protease activity digested she ep immunoglobulin heavy chain and bovine serum albumin but not bovine hemoglobin. Amino-terminal protein sequence analysis of the 29-kDa NEJ protease band revealed two sequences with homology to the cathepsin B family of proteases. Using degenerate oligonucleotides designed from the N-terminal sequence, reverse transcriptase polymerase chain reacti on with NEJ RNA amplified a cDNA sequence encoding the first 236 amino acids of mature cathepsin B. Using this cDNA fragment an overlapping cDNA was isolated from a LambdaZAP cDNA library constructed with poly( A)(+) RNA from immature 5-week-old liver fluke. Together with the N-te rminal sequence, these cDNAs predict a mature cathepsin B sequence of 254 amino acids which shows 48-51% sequence identity to mammalian and Schistosoma mansoni cathepsin B. We conclude that, in contrast to the major proteases released by adult fluke, the major secreted protease o f NEJ of F. hepatica is of the cathepsin B class. (C) 1998 Academic Pr ess.