Authors

D Smith¹; I Tikhonova¹; O C Drysdale¹; J Dvorak¹; M W Robinson¹; K Cwiklinski¹; J P Dalton¹;  ¹ Queen's University Belfast

Discussion

Kunitz-type (KT) protease inhibitors are low molecular weight proteins classically defined as serine protease inhibitors. A KT inhibitor (rFhKT1) is a major protein secreted by Fasciola hepatica during the infective juvenile stage. Unexpectedly, rFhKT1 exhibited no inhibitory activity towards serine proteases but was a potent inhibitor of the major secreted cathepsin L cysteine proteases of F. hepatica, FhCL1 and FhCL2, and of human cathepsins L and K (Ki = 0.24 - 25.607 nM). rFhKT1 prevented autocatalytic activation of FhCL1 and FhCL2 and formed stable complexes with the mature enzymes. Pull-down experiments showed that rFhKT1 interacts specifically with native secreted FhCL1, FhCL2 and FhCL5. Substitution of the unusual P1 Leu15 within the exposed reactive loop of FhKT1 for the more commonly found Arg had modest adverse effects on cysteine protease inhibition but conferred potent activity against the serine protease trypsin (Ki = 2.28 nM). Computational docking and sequence analysis demonstrated the importance of Leu15 in anchoring the inhibitor into the S2-S3 active site pocket, conferring selectivity towards cathepsin L-like proteases.  FhKT1 represents a novel evolutionary adaptation of KT protease inhibitors by F. hepatica, with its prime purpose likely in the regulation of the major parasite-secreted proteases and/or host proteases making this a novel vaccine and drug target.