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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.


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rFhKT1 inhibits mature FhCL1 and forms a stable complex.A, progress curve of fully activated FhCL1 inhibited by rFhKT1 over 360 min. Activity is represented by relative fluorescent units. Activity of uninhibited FhCL1 is represented by the solid line, and rFhKT1-inhibited FhCL1 is represented by the dashed line. Error bars indicate standard deviation of three separate experiments. B, FhCL1 zymogen (lane 1) was auto-catalytically activated at pH 4.5 for 1 h to the mature active enzyme (lane 2). The mature enzyme was then incubated in the absence (lanes 3–5) or presence (lanes 6–8) of rFhKT1 for 1 h (lanes 3 and 6), 2 h (lanes 4 and 7), and 6 h (lanes 5 and 8), and then the reaction mixes were analyzed by LDS-PAGE. The protein gel was stained with Biosafe Coomassie (Bio-Rad). Replicate gels were used for Western blot analysis by probing with polyclonal anti-rFhKT1 (C) and polyclonal antibody to His6-tagged proteins polyclonal antibodies (D). The arrowhead indicates the position of a cleaved product of ∼5-kDa band that reacts with polyclonal anti-rFhKT1 but not anti-His6-tagged antibodies indicating that it represents a cleavage product of rFhKT1. M, molecular mass markers.
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Figure 6: rFhKT1 inhibits mature FhCL1 and forms a stable complex.A, progress curve of fully activated FhCL1 inhibited by rFhKT1 over 360 min. Activity is represented by relative fluorescent units. Activity of uninhibited FhCL1 is represented by the solid line, and rFhKT1-inhibited FhCL1 is represented by the dashed line. Error bars indicate standard deviation of three separate experiments. B, FhCL1 zymogen (lane 1) was auto-catalytically activated at pH 4.5 for 1 h to the mature active enzyme (lane 2). The mature enzyme was then incubated in the absence (lanes 3–5) or presence (lanes 6–8) of rFhKT1 for 1 h (lanes 3 and 6), 2 h (lanes 4 and 7), and 6 h (lanes 5 and 8), and then the reaction mixes were analyzed by LDS-PAGE. The protein gel was stained with Biosafe Coomassie (Bio-Rad). Replicate gels were used for Western blot analysis by probing with polyclonal anti-rFhKT1 (C) and polyclonal antibody to His6-tagged proteins polyclonal antibodies (D). The arrowhead indicates the position of a cleaved product of ∼5-kDa band that reacts with polyclonal anti-rFhKT1 but not anti-His6-tagged antibodies indicating that it represents a cleavage product of rFhKT1. M, molecular mass markers.

Mentions: To examine the stability of inactivation of FhCL1 by rFhKT1 over time, FhCL1 was first auto-catalytically activated at pH 4.5 and then incubated with rFhKT1 for 6 h at 37 °C in citrate phosphate buffer, pH 5.5, in the presence of substrate; the reaction was monitored by the release of the fluorophore (7-amino-4-methylcoumarin), and samples of the reactions were analyzed by LDS-PAGE (Fig. 6). rFhKT1 completely inactivated the FhCL1, and this inactivation was stable for the entire 6 h (Fig. 6A). Furthermore, the rFhKT1 remained intact over this time and was observed as a band at ∼6 kDa. However, a minor band at ∼5 kDa was observed at the 2- and 6-h time points (Fig. 6B). This band was not immunoreactive with anti-His tag monoclonal antibodies (Fig. 6D), but it was reactive with anti-rFhKT1 polyclonal antibodies (Fig. 6C), showing that this protein results from proteolytic clipping at the C terminus of rFhKT1 by FhCL1. A similar cleavage pattern was observed with rFhKT1Leu15/Arg15 and also when FhCL2 was used in the reactions (data not shown).


Unexpected Activity of a Novel Kunitz-type Inhibitor
rFhKT1 inhibits mature FhCL1 and forms a stable complex.A, progress curve of fully activated FhCL1 inhibited by rFhKT1 over 360 min. Activity is represented by relative fluorescent units. Activity of uninhibited FhCL1 is represented by the solid line, and rFhKT1-inhibited FhCL1 is represented by the dashed line. Error bars indicate standard deviation of three separate experiments. B, FhCL1 zymogen (lane 1) was auto-catalytically activated at pH 4.5 for 1 h to the mature active enzyme (lane 2). The mature enzyme was then incubated in the absence (lanes 3–5) or presence (lanes 6–8) of rFhKT1 for 1 h (lanes 3 and 6), 2 h (lanes 4 and 7), and 6 h (lanes 5 and 8), and then the reaction mixes were analyzed by LDS-PAGE. The protein gel was stained with Biosafe Coomassie (Bio-Rad). Replicate gels were used for Western blot analysis by probing with polyclonal anti-rFhKT1 (C) and polyclonal antibody to His6-tagged proteins polyclonal antibodies (D). The arrowhead indicates the position of a cleaved product of ∼5-kDa band that reacts with polyclonal anti-rFhKT1 but not anti-His6-tagged antibodies indicating that it represents a cleavage product of rFhKT1. M, molecular mass markers.
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Figure 6: rFhKT1 inhibits mature FhCL1 and forms a stable complex.A, progress curve of fully activated FhCL1 inhibited by rFhKT1 over 360 min. Activity is represented by relative fluorescent units. Activity of uninhibited FhCL1 is represented by the solid line, and rFhKT1-inhibited FhCL1 is represented by the dashed line. Error bars indicate standard deviation of three separate experiments. B, FhCL1 zymogen (lane 1) was auto-catalytically activated at pH 4.5 for 1 h to the mature active enzyme (lane 2). The mature enzyme was then incubated in the absence (lanes 3–5) or presence (lanes 6–8) of rFhKT1 for 1 h (lanes 3 and 6), 2 h (lanes 4 and 7), and 6 h (lanes 5 and 8), and then the reaction mixes were analyzed by LDS-PAGE. The protein gel was stained with Biosafe Coomassie (Bio-Rad). Replicate gels were used for Western blot analysis by probing with polyclonal anti-rFhKT1 (C) and polyclonal antibody to His6-tagged proteins polyclonal antibodies (D). The arrowhead indicates the position of a cleaved product of ∼5-kDa band that reacts with polyclonal anti-rFhKT1 but not anti-His6-tagged antibodies indicating that it represents a cleavage product of rFhKT1. M, molecular mass markers.
Mentions: To examine the stability of inactivation of FhCL1 by rFhKT1 over time, FhCL1 was first auto-catalytically activated at pH 4.5 and then incubated with rFhKT1 for 6 h at 37 °C in citrate phosphate buffer, pH 5.5, in the presence of substrate; the reaction was monitored by the release of the fluorophore (7-amino-4-methylcoumarin), and samples of the reactions were analyzed by LDS-PAGE (Fig. 6). rFhKT1 completely inactivated the FhCL1, and this inactivation was stable for the entire 6 h (Fig. 6A). Furthermore, the rFhKT1 remained intact over this time and was observed as a band at ∼6 kDa. However, a minor band at ∼5 kDa was observed at the 2- and 6-h time points (Fig. 6B). This band was not immunoreactive with anti-His tag monoclonal antibodies (Fig. 6D), but it was reactive with anti-rFhKT1 polyclonal antibodies (Fig. 6C), showing that this protein results from proteolytic clipping at the C terminus of rFhKT1 by FhCL1. A similar cleavage pattern was observed with rFhKT1Leu15/Arg15 and also when FhCL2 was used in the reactions (data not shown).

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.


Related in: MedlinePlus