A single amino acid substitution affects substrate specificity in cysteineproteinases from Fasciola hepatica

The trematode Fasciola hepatica secretes a number of cathepsin L-like prote ases that are proposed to be involved in feeding, migration, and immune eva sion by the parasite. To date, six full cDNA sequences encoding cathepsin L preproproteins have been identified. Previous studies have demonstrated th at one of these cathepsins (L2) is unusual in that it is able to cleave sub strates with a proline in the P-2 position, translating into an unusual abi lity (for a cysteine proteinase) to clot fibrinogen. In this study, we repo rt the sequence of a novel cathepsin (L5) and compare the substrate specifi city of a recombinant enzyme with that of recombinant cathepsin L2. Despite sharing 80% sequence identity with cathepsin L2, cathepsin L5 does not exh ibit substantial catalytic activity against substrates containing proline i n the P-2 position. Molecular modeling studies suggested that a single amin o acid change (L69Y) in the mature proteinases may account for the differen ce in specificity at the S-2 subsite. Recombinant cathepsin L5/L69Y was exp ressed in yeast and a substantial increase in the ability of this variant t o accommodate substrates with a proline residue in the P-2 position was obs erved. Thus, we have identified a single amino acid substitution that can s ubstantially influence the architecture of the S-2 subsite of F. hepatica c athepsin L proteases.