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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.


FhKT1 is a temporally regulated and secreted protein in F. hepatica NEJ.A, graphical representation of fhkt1 expression within NEJs 3 and 24 h post-excystment in transcripts per kilobase million (TPM). Error bars indicate standard deviation of two separate experiments. B, Western blot analysis of RPMI 1640 culture medium from F. hepatica NEJs cultured for 24 h. Following the transfer of protein from LDS-polyacrylamide gel onto a nitrocellulose membrane by Western blotting, blots were spliced and probed with pre-immune mouse antiserum (lane 1) and anti-FhKT1 monoclonal antibodies raised in mice (lane 2). The immunoreactive band in lane 2 corresponding to FhKT1 is indicated by a gray arrow. M, molecular mass marker. C, graphical representation of mass spectrometry analysis of FhKT1 present in RPMI 1640 culture medium from F. hepatica NEJs cultured for 3 and 24 h, shown as unique peptide number. Error bars indicate standard deviation of three separate experiments. D, immunolocalization of FhKT1 in NEJs by confocal scanning laser microscopy. NEJs were fixed and stained as described under “Experimental Procedures.” FhKT1 expression in NEJs from two time points were compared, 3 h post-excystment (panels i–iii) and 24 h post-excystment (panels iv–vi). Fixed NEJs were probed with either anti-FhKT1 polyclonal antibodies raised in rabbit (panels ii, iii, v, and vi) or rabbit pre-immune antiserum (negative controls, panels i and iv), followed by the secondary antibody, fluorescein isothiocyanate (FITC)-labeled goat anti-rabbit IgG. No FITC staining was observed in the negative controls (panels i and v). Very little FITC staining (green fluorescence) was evident in the 3-h NEJs (panels ii and iii). FITC staining was observed in 24-h NEJs (panels v and vi), with FhKT1 being evident in the parenchyma (particularly concentrated in parenchymal cell bodies, white arrowheads) and gut (black arrowheads). Panels iii and vi and high power images of panels ii and v, respectively. All specimens were counter-stained with phalloidin-tetramethylrhodamine isothiocyanate (TRITC) to stain muscle tissue (red fluorescence) and provide structure. OS, oral sucker; VS, ventral sucker. Scale bars, 20 μm. M, molecular mass markers.
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Figure 1: FhKT1 is a temporally regulated and secreted protein in F. hepatica NEJ.A, graphical representation of fhkt1 expression within NEJs 3 and 24 h post-excystment in transcripts per kilobase million (TPM). Error bars indicate standard deviation of two separate experiments. B, Western blot analysis of RPMI 1640 culture medium from F. hepatica NEJs cultured for 24 h. Following the transfer of protein from LDS-polyacrylamide gel onto a nitrocellulose membrane by Western blotting, blots were spliced and probed with pre-immune mouse antiserum (lane 1) and anti-FhKT1 monoclonal antibodies raised in mice (lane 2). The immunoreactive band in lane 2 corresponding to FhKT1 is indicated by a gray arrow. M, molecular mass marker. C, graphical representation of mass spectrometry analysis of FhKT1 present in RPMI 1640 culture medium from F. hepatica NEJs cultured for 3 and 24 h, shown as unique peptide number. Error bars indicate standard deviation of three separate experiments. D, immunolocalization of FhKT1 in NEJs by confocal scanning laser microscopy. NEJs were fixed and stained as described under “Experimental Procedures.” FhKT1 expression in NEJs from two time points were compared, 3 h post-excystment (panels i–iii) and 24 h post-excystment (panels iv–vi). Fixed NEJs were probed with either anti-FhKT1 polyclonal antibodies raised in rabbit (panels ii, iii, v, and vi) or rabbit pre-immune antiserum (negative controls, panels i and iv), followed by the secondary antibody, fluorescein isothiocyanate (FITC)-labeled goat anti-rabbit IgG. No FITC staining was observed in the negative controls (panels i and v). Very little FITC staining (green fluorescence) was evident in the 3-h NEJs (panels ii and iii). FITC staining was observed in 24-h NEJs (panels v and vi), with FhKT1 being evident in the parenchyma (particularly concentrated in parenchymal cell bodies, white arrowheads) and gut (black arrowheads). Panels iii and vi and high power images of panels ii and v, respectively. All specimens were counter-stained with phalloidin-tetramethylrhodamine isothiocyanate (TRITC) to stain muscle tissue (red fluorescence) and provide structure. OS, oral sucker; VS, ventral sucker. Scale bars, 20 μm. M, molecular mass markers.

Mentions: Transcriptome analysis of the newly excysted juvenile stage showed that fhkt1 is expressed by the early F. hepatica stages that invade the host. Differential gene expression analysis during the first 24 h post-excystment shows that fhkt1 gene is up-regulated in NEJs 24 h post-excystment compared with NEJs 3 h post-excystment (Fig. 1A). Western blot analysis using anti-FhKT1 antibodies revealed an ∼7-kDa band in medium in which NEJs were cultured for 24 h (Fig. 1B, lane 2), confirming that FhKT1 is secreted by this parasite the 1st day after excystment. Mass spectrometry analysis of NEJ-secreted products revealed that FhKT1 is released into medium by both 3- and 24-h post-excystment NEJs (Fig. 1C and supplemental file 1).


Unexpected Activity of a Novel Kunitz-type Inhibitor
FhKT1 is a temporally regulated and secreted protein in F. hepatica NEJ.A, graphical representation of fhkt1 expression within NEJs 3 and 24 h post-excystment in transcripts per kilobase million (TPM). Error bars indicate standard deviation of two separate experiments. B, Western blot analysis of RPMI 1640 culture medium from F. hepatica NEJs cultured for 24 h. Following the transfer of protein from LDS-polyacrylamide gel onto a nitrocellulose membrane by Western blotting, blots were spliced and probed with pre-immune mouse antiserum (lane 1) and anti-FhKT1 monoclonal antibodies raised in mice (lane 2). The immunoreactive band in lane 2 corresponding to FhKT1 is indicated by a gray arrow. M, molecular mass marker. C, graphical representation of mass spectrometry analysis of FhKT1 present in RPMI 1640 culture medium from F. hepatica NEJs cultured for 3 and 24 h, shown as unique peptide number. Error bars indicate standard deviation of three separate experiments. D, immunolocalization of FhKT1 in NEJs by confocal scanning laser microscopy. NEJs were fixed and stained as described under “Experimental Procedures.” FhKT1 expression in NEJs from two time points were compared, 3 h post-excystment (panels i–iii) and 24 h post-excystment (panels iv–vi). Fixed NEJs were probed with either anti-FhKT1 polyclonal antibodies raised in rabbit (panels ii, iii, v, and vi) or rabbit pre-immune antiserum (negative controls, panels i and iv), followed by the secondary antibody, fluorescein isothiocyanate (FITC)-labeled goat anti-rabbit IgG. No FITC staining was observed in the negative controls (panels i and v). Very little FITC staining (green fluorescence) was evident in the 3-h NEJs (panels ii and iii). FITC staining was observed in 24-h NEJs (panels v and vi), with FhKT1 being evident in the parenchyma (particularly concentrated in parenchymal cell bodies, white arrowheads) and gut (black arrowheads). Panels iii and vi and high power images of panels ii and v, respectively. All specimens were counter-stained with phalloidin-tetramethylrhodamine isothiocyanate (TRITC) to stain muscle tissue (red fluorescence) and provide structure. OS, oral sucker; VS, ventral sucker. Scale bars, 20 μm. M, molecular mass markers.
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Figure 1: FhKT1 is a temporally regulated and secreted protein in F. hepatica NEJ.A, graphical representation of fhkt1 expression within NEJs 3 and 24 h post-excystment in transcripts per kilobase million (TPM). Error bars indicate standard deviation of two separate experiments. B, Western blot analysis of RPMI 1640 culture medium from F. hepatica NEJs cultured for 24 h. Following the transfer of protein from LDS-polyacrylamide gel onto a nitrocellulose membrane by Western blotting, blots were spliced and probed with pre-immune mouse antiserum (lane 1) and anti-FhKT1 monoclonal antibodies raised in mice (lane 2). The immunoreactive band in lane 2 corresponding to FhKT1 is indicated by a gray arrow. M, molecular mass marker. C, graphical representation of mass spectrometry analysis of FhKT1 present in RPMI 1640 culture medium from F. hepatica NEJs cultured for 3 and 24 h, shown as unique peptide number. Error bars indicate standard deviation of three separate experiments. D, immunolocalization of FhKT1 in NEJs by confocal scanning laser microscopy. NEJs were fixed and stained as described under “Experimental Procedures.” FhKT1 expression in NEJs from two time points were compared, 3 h post-excystment (panels i–iii) and 24 h post-excystment (panels iv–vi). Fixed NEJs were probed with either anti-FhKT1 polyclonal antibodies raised in rabbit (panels ii, iii, v, and vi) or rabbit pre-immune antiserum (negative controls, panels i and iv), followed by the secondary antibody, fluorescein isothiocyanate (FITC)-labeled goat anti-rabbit IgG. No FITC staining was observed in the negative controls (panels i and v). Very little FITC staining (green fluorescence) was evident in the 3-h NEJs (panels ii and iii). FITC staining was observed in 24-h NEJs (panels v and vi), with FhKT1 being evident in the parenchyma (particularly concentrated in parenchymal cell bodies, white arrowheads) and gut (black arrowheads). Panels iii and vi and high power images of panels ii and v, respectively. All specimens were counter-stained with phalloidin-tetramethylrhodamine isothiocyanate (TRITC) to stain muscle tissue (red fluorescence) and provide structure. OS, oral sucker; VS, ventral sucker. Scale bars, 20 μm. M, molecular mass markers.
Mentions: Transcriptome analysis of the newly excysted juvenile stage showed that fhkt1 is expressed by the early F. hepatica stages that invade the host. Differential gene expression analysis during the first 24 h post-excystment shows that fhkt1 gene is up-regulated in NEJs 24 h post-excystment compared with NEJs 3 h post-excystment (Fig. 1A). Western blot analysis using anti-FhKT1 antibodies revealed an ∼7-kDa band in medium in which NEJs were cultured for 24 h (Fig. 1B, lane 2), confirming that FhKT1 is secreted by this parasite the 1st day after excystment. Mass spectrometry analysis of NEJ-secreted products revealed that FhKT1 is released into medium by both 3- and 24-h post-excystment NEJs (Fig. 1C and supplemental file 1).

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.