Unexpected Activity of a Novel Kunitz-type Inhibitor

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.

Fasciola hepatica-Derived Molecules as Regulators of the Host Immune Response

“…Kunitz serine protease inhibitors have been identified in the total extract and tegument of F. hepatica ( 54 ). Interestingly, F. hepatica Kunitz type molecule (FhKTM) has an unique specificity for cysteine proteases ( 13 ) and was shown to associate with cathepsin L ( 55 ). FhKTM induced a regulatory IL-27-dependent phenotype in LPS-stimulated DCs that impaired Th1 and Th17 responses ( 56 ).…”

Functional diversity of secreted cestode Kunitz proteins: Inhibition of serine peptidases and blockade of cation channels

“…As to the other groupings, EgrG_001136500 (Leu in P1) was recently found to be a potent inhibitor of neutrophil elastase ( K I ~ 10 −11 M) and cathepsin G ( K I ~ 10 −10 M) and, interestingly, to reduce neutrophil infiltration in a local inflammation model (EgKI-2 in [ 36 ]); thus, its close paralog (EgrG_00113800; Arg in P1) could also be a serine peptidase inhibitor. Finally, the sequences from Fasciola hepatica (FhKTM [ 42 ] and FhKT1 [ 43 ], both with Leu in P1, whose Kunitz domains differ in 3/51 amino acids) define a basal, separate sub-clade, that could also reflect functional diversity: FhKTM was found to be a marginal inhibitor of trypsin with virtually no effect over chymotrypsin [ 42 ] but, notably, FhKT1 was recently characterized as an inhibitor of cysteine peptidases, including the major parasite cathepsin L secreted peptidases and related human peptidases [ 43 ]).…”

“…The sequences from F . hepatica (FhKTM [ 42 ] and FhKT1 [ 43 ]) define a basal, separate clade that could reflect functional diversity (cysteine peptidase inhibition; [ 43 ]). The long branch of Eg KU-2 (and its putative T .…”

“…Furthermore, a similar result was observed with the Kunitz protein FhKTM from the trematode F . hepatica , which is known to be present in parasite E/S products [ 57 ].As already mentioned, FhKTM showed a marginal serine peptidase inhibitory activity [ 42 ] but, notably, the very closely related FhKT1 (> 90% overall identity with FhKTM) was recently found to inhibit cysteine peptidases [ 43 ].…”

“…solium genome project [ 5 ] (encoded by genes TsM_000321400, TsM_000410200, TsM_000513000, TsM_000576900, TsM_000647700, TsM_000724900, TsM_001022000, TsM_001027800, TsM_001085400, TsM_001162600 retrieved from GeneDB— http://www.genedb.org/Homepage/Tsolium ; and the EST EL746785 retrieved as TSE0004567 from PartiGeneDB— http://www.compsysbio.org/partigene/ ) were added to this set. A group of five functionally annotated sequences from other Lophotrochozoa was also included: FhKTM (UniProt Q9TXD3; [ 42 ]) and FhKT1 [ 43 ], from F . hepatica ; SjKI-1 (Sjp_0020270) from S .…”