Unexpected Activity of a Novel Kunitz-type Inhibitor: INHIBITION OF CYSTEINE PROTEASES BUT NOT SERINE PROTEASES.

Smith, David ORCID: 0000-0002-5158-0522, Tikhonova, Irina G, Jewhurst, Heather L, Drysdale, Orla C, Dvořák, Jan ORCID: 0000-0002-6824-9000, Robinson, Mark W ORCID: 0000-0001-7191-0034, Cwiklinski, Krystyna ORCID: 0000-0001-5577-2735 and Dalton, John P (2016) Unexpected Activity of a Novel Kunitz-type Inhibitor: INHIBITION OF CYSTEINE PROTEASES BUT NOT SERINE PROTEASES. The Journal of biological chemistry, 291 (37). pp. 19220-19234.

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Official URL: http://dx.doi.org/10.1074/jbc.m116.724344

Abstract

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 (Ki = 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 (Ki = 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.

Item Type:	Article
Uncontrolled Keywords:	Animals, Humans, Fasciola hepatica, Trypsin, Helminth Proteins, Recombinant Proteins, Cysteine Proteinase Inhibitors, Trypsin Inhibitors, Cathepsin K, Cathepsin L
Divisions:	Faculty of Health and Life Sciences Faculty of Health and Life Sciences > Institute of Infection, Veterinary and Ecological Sciences
Depositing User:	Symplectic Admin
Date Deposited:	24 Feb 2022 09:10
Last Modified:	14 Mar 2024 17:46
DOI:	10.1074/jbc.m116.724344
Open Access URL:	https://www.sciencedirect.com/science/article/pii/...
Related URLs:	Author Publisher
URI:	https://livrepository.liverpool.ac.uk/id/eprint/3149585