The major secreted cathepsin L1 protease of the liver fluke, Fasciola hepatica: A Leu^-12 to Pro^-12 replacement in the nonconserved C-terminal region of the prosegment prevents complete enzyme autoactivation and allows definition of the molecular events in prosegment removal

A protease secreted by the parasitic helminth Fasciola hepatica, a 37-kDa procathepsin L1 (FheproCL1), autocatalytically processes and activates to its mature enzyme (FheCL1) over a wide pH range of 7.3 to 4.0, although activation is more rapid at low pH. Maturation initiates with cleavages of a small proportion of molecules within the central region of the prosegment, possibly by intramolecular events. However, activation to fully mature enzymes is achieved by a precise intermolecular cleavage at a Leu^-12-Ser ^-11↓His^-10 sequence within the nonconserved C-terminal region of the prosegment. The importance of this cleavage site in enzyme activation was demonstrated using an active site variant FheproCL1Gly²⁶ (Cys²⁶ to Gly²⁶) and a double variant FheproCL1Pro^-12/Gly²⁶ (Leu^-12 to Pro^-12), and although both of these variants cannot autocatalytically process, the former is susceptible to trans-processing at a Leu ^-12-Ser^-11↓His^-10 sequence by pre-activated FheCL1, but the latter is not. Another F. hepatica secreted protease FheCL2, which, unlike FheCL1, can readily accept proline in the S2 subsite of its active site, can trans-process the double variant FheproCL1Pro^-12/Gly²⁶ by cleavage at the Pro ^-12-Ser^-11↓His^-10 sequence. Furthermore, the autoactivation of a variant enzyme with a single replacement, FheproCL1Pro^-12, was very slow but was increased 40-fold in the presence of FheCL2. These studies provide a molecular insight into the regulation of FheproCL1 autocatalysis.