Journal
JOURNAL OF BIOLOGICAL CHEMISTRY
Volume 291, Issue 37, Pages 19220-19234Publisher
ELSEVIER
DOI: 10.1074/jbc.M116.724344
Keywords
cysteine protease; enzyme inhibitor; enzyme kinetics; host-pathogen interaction; molecular modeling; parasite; protease inhibitor; protein-protein interaction; serine protease
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Funding
- Department for Employment and Learning (DEL), Northern Ireland grant
- European Research Council Advanced Grant HELIVAC [European Research Council Advanced Grant HELIVAC 322725]
- Biotechnology and Biological Sciences Research Council (BBSRC) [BB/L019612/1]
- Biotechnology and Biological Sciences Research Council [BB/L019612/1] Funding Source: researchfish
- BBSRC [BB/L019612/1] Funding Source: UKRI
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Kunitz-type (KT) protease inhibitors are low molecular weight proteins classically defined as serine protease inhibitors. We identified a novel secreted KT inhibitor associated with the gut and parenchymal tissues of the infective juvenile stage of Fasciola hepatica, a helminth parasite of medical and veterinary importance. Unexpectedly, recombinant KT inhibitor (rFhKT1) exhibited no inhibitory activity toward 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 (K-i = 0.4-27 nm). FhKT1 prevented the auto-catalytic activation of FhCL1 and FhCL2 and formed stable complexes with the mature enzymes. Pulldown experiments from adult parasite culture medium showed that rFhKT1 interacts specifically with native secreted FhCL1, FhCL2, and FhCL5. Substitution of the unusual P1 Leu(15) within the exposed reactive loop of FhKT1 for the more commonly found Arg (FhKT1Leu(15)/Arg(15)) had modest adverse effects on the cysteine protease inhibition but conferred potent activity against the serine protease trypsin (K-i = 1.5 nm). Computational docking and sequence analysis provided hypotheses for the exclusive binding of FhKT1 to cysteine proteases, the importance of the Leu(15) in anchoring the inhibitor into the S2 active site pocket, and the inhibitor's selectivity toward FhCL1, FhCL2, and human cathepsins L and K. 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 cathepsin L-like proteases of its host.
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