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Unexpected Activity of a Novel Kunitz-type Inhibitor

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.


Inhibitory activity of rFhKT1 and rFhKT1Leu15/Arg15 is dependent on disulfide bridges.A, inhibition of FhCL1 activity by rFhKT1 and rFhKT1Leu15/Arg15 before (black bars) and after reduction (R) and alkylation (A) (light gray bars) and with reduced and alkylated inhibitor following incubation for 10 days at 4 °C (dark gray bars). B, inhibition of trypsin activity by rFhKT1Leu15/Arg15 before (pre-reduction and alkylation (R/A), black bars) and after reduction and alkylation (R/A, light gray bars), and with reduced and alkylated inhibitor following incubation for 10 days at 4 °C (post-R/A, dark gray bars). Error bars indicate standard deviation.
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Figure 7: Inhibitory activity of rFhKT1 and rFhKT1Leu15/Arg15 is dependent on disulfide bridges.A, inhibition of FhCL1 activity by rFhKT1 and rFhKT1Leu15/Arg15 before (black bars) and after reduction (R) and alkylation (A) (light gray bars) and with reduced and alkylated inhibitor following incubation for 10 days at 4 °C (dark gray bars). B, inhibition of trypsin activity by rFhKT1Leu15/Arg15 before (pre-reduction and alkylation (R/A), black bars) and after reduction and alkylation (R/A, light gray bars), and with reduced and alkylated inhibitor following incubation for 10 days at 4 °C (post-R/A, dark gray bars). Error bars indicate standard deviation.

Mentions: Reduction and alkylation of both rFhKT1 and rFhKT1Leu15/Arg15 were performed to break internal disulfide bridges and to disrupt the tertiary structure of the protein. This procedure depleted the inhibitory activity of both inhibitors against FhCL1 (Fig. 7A). Similarly, this treatment ablated the inhibitory activity of rFhKT1Leu15/Arg15 against trypsin (Fig. 7B). Interestingly, we discovered that both rFhKT1 and rFhKT1Leu15/Arg15 could partially re-nature to their functional form during a 10-day incubation period at 4 °C; over this time, the inhibitors regained between 60 and 80% of their inhibitory activity (Fig. 7A). Likewise, rFhKT1Leu15/Arg15 regained inhibitory activity against trypsin (Fig. 7B).


Unexpected Activity of a Novel Kunitz-type Inhibitor
Inhibitory activity of rFhKT1 and rFhKT1Leu15/Arg15 is dependent on disulfide bridges.A, inhibition of FhCL1 activity by rFhKT1 and rFhKT1Leu15/Arg15 before (black bars) and after reduction (R) and alkylation (A) (light gray bars) and with reduced and alkylated inhibitor following incubation for 10 days at 4 °C (dark gray bars). B, inhibition of trypsin activity by rFhKT1Leu15/Arg15 before (pre-reduction and alkylation (R/A), black bars) and after reduction and alkylation (R/A, light gray bars), and with reduced and alkylated inhibitor following incubation for 10 days at 4 °C (post-R/A, dark gray bars). Error bars indicate standard deviation.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5016662&req=5

Figure 7: Inhibitory activity of rFhKT1 and rFhKT1Leu15/Arg15 is dependent on disulfide bridges.A, inhibition of FhCL1 activity by rFhKT1 and rFhKT1Leu15/Arg15 before (black bars) and after reduction (R) and alkylation (A) (light gray bars) and with reduced and alkylated inhibitor following incubation for 10 days at 4 °C (dark gray bars). B, inhibition of trypsin activity by rFhKT1Leu15/Arg15 before (pre-reduction and alkylation (R/A), black bars) and after reduction and alkylation (R/A, light gray bars), and with reduced and alkylated inhibitor following incubation for 10 days at 4 °C (post-R/A, dark gray bars). Error bars indicate standard deviation.
Mentions: Reduction and alkylation of both rFhKT1 and rFhKT1Leu15/Arg15 were performed to break internal disulfide bridges and to disrupt the tertiary structure of the protein. This procedure depleted the inhibitory activity of both inhibitors against FhCL1 (Fig. 7A). Similarly, this treatment ablated the inhibitory activity of rFhKT1Leu15/Arg15 against trypsin (Fig. 7B). Interestingly, we discovered that both rFhKT1 and rFhKT1Leu15/Arg15 could partially re-nature to their functional form during a 10-day incubation period at 4 °C; over this time, the inhibitors regained between 60 and 80% of their inhibitory activity (Fig. 7A). Likewise, rFhKT1Leu15/Arg15 regained inhibitory activity against trypsin (Fig. 7B).

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.