The trematode Fasciola hepatica secretes
a number of cathepsin L-like proteases that are proposed
to be involved in feeding, migration, and immune evasion
by the parasite. To date, six full cDNA sequences encoding
cathepsin L preproproteins have been identified. Previous
studies have demonstrated that one of these cathepsins
(L2) is unusual in that it is able to cleave substrates
with a proline in the P2 position, translating
into an unusual ability (for a cysteine proteinase) to
clot fibrinogen. In this study, we report the sequence
of a novel cathepsin (L5) and compare the substrate specificity
of a recombinant enzyme with that of recombinant cathepsin
L2. Despite sharing 80% sequence identity with cathepsin
L2, cathepsin L5 does not exhibit substantial catalytic
activity against substrates containing proline in the P2
position. Molecular modeling studies suggested that a single
amino acid change (L69Y) in the mature proteinases may
account for the difference in specificity at the S2
subsite. Recombinant cathepsin L5/L69Y was expressed in
yeast and a substantial increase in the ability of this
variant to accommodate substrates with a proline residue
in the P2 position was observed. Thus, we have
identified a single amino acid substitution that can substantially
influence the architecture of the S2 subsite
of F. hepatica cathepsin L proteases.