Cloning, yeast expression, mutagenesis and phylogenetic analysis of a novel member of the Fasciola hepatica cathepsin L-like family

Cathepsin L2, a major cysteine proteinase secreted by adult Fasciola hepatica, differs from other reported cathepsin L-like enzymes in its’ ability to cleave peptide substrates that contain proline in the P2 position. In the present study we have isolated a cDNA clone encoding a complete cysteine proteinase precursor from a Fasciola hepatica cDNA library screened with anti-cathepsin L2 serum. The deduced amino acid sequence was compared with other cysteine proteinases of F. hepatica. This confirmed that it belongs to a gene family composed of at least five different cathepsin L-like genes, and is different from other F. hepatica secreted cathepsin L-like proteinases. The cloned gene was successfully expressed in yeast using the trafficking signals contained within its own propeptide, resulting in functionally active enzyme. Comparison of the yeast expressed enzyme and native liver fluke cathepsin L2 by immunological and biochemical analysis showed that the cloned zymogen encoded for the liver fluke cathepsin L2. Cathepsin L2 differs in substrate specificity from F. hepatica cathepsin LI. To test if this difference is due to a tyrosine in the active site, site directed mutagenesis was performed to convert the leucine present in cathepsin LI to the tyrosine present in cathepsin L2. The data obtained indicate that this substitution is not directly linked to the differences in substrate specificity observed between liver fluke cathepsin LI and cathepsin L2. The mutated purified enzyme was not capable of cleaving substrates with proline in the P2 position. Phylogenetic analysis of the papain superfamily indicated that at least four different types of cysteine proteinases of the papain superfamily exist in trematodes. The liver fluke enzymes cloned so far, constitute a cysteine proteinase family equally related to the vertebrate cathepsin Ls, cathepsin Ss and cathepsin Ks. Other relationships between cysteine proteinases of diverse origin were also detected, which allowed us to group them into families and classes