Unexpected Activity of a Novel Kunitz-type Inhibitor

Content Provider	Scilit
Author	Smith, David Tikhonova, Irina G. Jewhurst, Heather L. Drysdale, Orla C. Dvořák, Jan Robinson, Mark W. Cwiklinski, Krystyna Dalton, John P.
Copyright Year	2016
Description	Journal: Journal of Biological Chemistry 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.
Related Links	http://www.jbc.org/content/291/37/19220.full.pdf https://core.ac.uk/download/pdf/74405058.pdf https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5016662/pdf http://www.jbc.org/article/S0021925820360038/pdf
Ending Page	19234
Page Count	15
Starting Page	19220
ISSN	00219258
e-ISSN	1083351X
DOI	10.1074/jbc.m116.724344
Journal	Journal of Biological Chemistry
Issue Number	37
Volume Number	291
Language	English
Publisher	Elsevier BV
Publisher Date	2016-09-01
Access Restriction	Open
Subject Keyword	Journal: Journal of Biological Chemistry Biochemistry and Molecular Biology Cysteine Protease Enzyme Inhibitor Enzyme Kinetics Host-pathogen Interaction Molecular Modeling Protease Inhibitor Protein-protein Interaction Serine Protease
Content Type	Text
Resource Type	Article
Subject	Cell Biology Biochemistry Molecular Biology