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 Leu15 within the exposed reactive loop of FhKT1 for the more commonly found Arg (FhKT1Leu15/Arg15) 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 Leu15 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.
Highlights
Inferior meat quality and parasite-related mortality, costing the agricultural industry an estimated United States $3 billion annually [1, 2]
We report the discovery of a KT protease inhibitor (FhKT1) from the infective and tissue invasive stage of F. hepatica that exhibits no inhibitory activity against serine proteases but has potent activity against cathepsin L-like cysteine proteases
Molecular modeling of the interaction of FhKT1 with the major secreted parasite cathepsin L cysteine proteases and human cathepsin L-like cysteine proteases indicated the importance of the P1 Leu15 in docking the inhibitor into the S2 active site of these enzymes and suggested key inhibitor-enzyme interactions that impart FhKT1 its unique inhibitory properties
Summary
Expression of fhkt in Newly Excysted Juveniles in F. hepatica—Transcriptome analysis of the newly excysted juvenile stage showed that fhkt is expressed by the early F. hepatica stages that invade the host. In our initial screens we found that the purified rFhKT1 did not exhibit any inhibition against a variety of standard serine proteases, including trypsin, chymotrypsin, elastase, thrombin, kallikrein, and human cathepsin G (Fig. 3A) This was not due to the recombinant protein being functionally inactive as we subsequently discovered that rFhKT1 was a potent inhibitor of two F. hepatica cathepsin L-like cysteine proteases, FhCL1 and FhCL2. Impact of Substituting the FhKT1 P1 Leu Residue to Arg on Protease Inhibition—Given the importance of the P1 residue at position 15 within the exposed loop in KT protease inhibitors, we examined the impact of substituting this residue in FhKT1 from a leucine to the more commonly found arginine This variant form, termed rFhKT1Leu15/Arg, retained its ability to inhibit F. hepatica FhCL1 and FhCL2 and human cathepsin L and K, but as with the wild-type rFhKT1, it did not inhibit FhCB1 and FhCB2 and human cathepsin B and S (Table 1). RFhKT1 and rFhKT1Leu15/Arg inhibition specificity against a panel of biologically relevant serine and cysteine proteases and relative inhibition constants rFhKT1
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