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Unexpected Activity of a Novel Kunitz-type Inhibitor

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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.

No MeSH data available.


Sequence alignment of F. hepatica and Homo sapiens cysteine proteases.F. hepatica cysteine protease sequences include FhCL1 (cathepsin L1, AAB41670), FhCL2 (cathepsin L2, AAC47721), FhCB1 (cathepsin B1, ABF85678), and FhCB2 (cathepsin B2, ABF85679). H. sapiens cysteine protease sequences include HsCK (cathepsin K, NP_000387), HsCL (cathepsin L, NP_001903), HsCS (cathepsin S, NP_004070), and HsCB (cathepsin B, NP_001899). Residues surrounding the S1 active site region are shown in green, and residues forming the S2 active site are shown in pink. Residues that form interactions with FhKT1 and FhKT1Leu15/Arg15 are shown in bold.
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Figure 10: Sequence alignment of F. hepatica and Homo sapiens cysteine proteases.F. hepatica cysteine protease sequences include FhCL1 (cathepsin L1, AAB41670), FhCL2 (cathepsin L2, AAC47721), FhCB1 (cathepsin B1, ABF85678), and FhCB2 (cathepsin B2, ABF85679). H. sapiens cysteine protease sequences include HsCK (cathepsin K, NP_000387), HsCL (cathepsin L, NP_001903), HsCS (cathepsin S, NP_004070), and HsCB (cathepsin B, NP_001899). Residues surrounding the S1 active site region are shown in green, and residues forming the S2 active site are shown in pink. Residues that form interactions with FhKT1 and FhKT1Leu15/Arg15 are shown in bold.

Mentions: In this model of interactions, the residue Leu15 of the exposed loop of FhKT1 forms strong hydrophobic interactions with residues Leu176, Met177, and Val267 of the S2 pocket of FhCL1 (Fig. 9, A and B). This places Gly16 and Gly17 across the S1 pocket, but neither penetrates this subsite. The Ile18 is not predicted to make any interactions with the FhCL1, but Arg19 forms an H-bond with the Asn128 of the S2′ pocket. Relevantly, the sequence alignment of the cysteine proteases (Tables 2 and 3 and Fig. 10) used in this study shows that residues of the S2 and S1′-S2′ pockets are highly similar in physicochemical properties in FhCL1, FhCL2, human cathepsins L and K, which are all inhibited by FhKT1. By contrast, these residues are diverse in F. hepatica cathepsins FhCB1 and FhCB2, and in human cathepsins B and S, which are not inhibited by FhKT1 (Tables 2 and 3 and Fig. 10). This observation suggests that the S2 and S1′-S2′ pockets play a crucial role in selective binding of FhKT1 to FhCL1, FhCL2, and human cathepsins L and K.


Unexpected Activity of a Novel Kunitz-type Inhibitor
Sequence alignment of F. hepatica and Homo sapiens cysteine proteases.F. hepatica cysteine protease sequences include FhCL1 (cathepsin L1, AAB41670), FhCL2 (cathepsin L2, AAC47721), FhCB1 (cathepsin B1, ABF85678), and FhCB2 (cathepsin B2, ABF85679). H. sapiens cysteine protease sequences include HsCK (cathepsin K, NP_000387), HsCL (cathepsin L, NP_001903), HsCS (cathepsin S, NP_004070), and HsCB (cathepsin B, NP_001899). Residues surrounding the S1 active site region are shown in green, and residues forming the S2 active site are shown in pink. Residues that form interactions with FhKT1 and FhKT1Leu15/Arg15 are shown in bold.
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Figure 10: Sequence alignment of F. hepatica and Homo sapiens cysteine proteases.F. hepatica cysteine protease sequences include FhCL1 (cathepsin L1, AAB41670), FhCL2 (cathepsin L2, AAC47721), FhCB1 (cathepsin B1, ABF85678), and FhCB2 (cathepsin B2, ABF85679). H. sapiens cysteine protease sequences include HsCK (cathepsin K, NP_000387), HsCL (cathepsin L, NP_001903), HsCS (cathepsin S, NP_004070), and HsCB (cathepsin B, NP_001899). Residues surrounding the S1 active site region are shown in green, and residues forming the S2 active site are shown in pink. Residues that form interactions with FhKT1 and FhKT1Leu15/Arg15 are shown in bold.
Mentions: In this model of interactions, the residue Leu15 of the exposed loop of FhKT1 forms strong hydrophobic interactions with residues Leu176, Met177, and Val267 of the S2 pocket of FhCL1 (Fig. 9, A and B). This places Gly16 and Gly17 across the S1 pocket, but neither penetrates this subsite. The Ile18 is not predicted to make any interactions with the FhCL1, but Arg19 forms an H-bond with the Asn128 of the S2′ pocket. Relevantly, the sequence alignment of the cysteine proteases (Tables 2 and 3 and Fig. 10) used in this study shows that residues of the S2 and S1′-S2′ pockets are highly similar in physicochemical properties in FhCL1, FhCL2, human cathepsins L and K, which are all inhibited by FhKT1. By contrast, these residues are diverse in F. hepatica cathepsins FhCB1 and FhCB2, and in human cathepsins B and S, which are not inhibited by FhKT1 (Tables 2 and 3 and Fig. 10). This observation suggests that the S2 and S1′-S2′ pockets play a crucial role in selective binding of FhKT1 to FhCL1, FhCL2, and human cathepsins L and K.

View Article: PubMed Central - PubMed

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.

No MeSH data available.