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Simukunin from the salivary glands of the black fly Simulium vittatum inhibits enzymes that regulate clotting and inflammatory responses.

Tsujimoto H, Kotsyfakis M, Francischetti IM, Eum JH, Strand MR, Champagne DE - PLoS ONE (2012)

Bottom Line: Our results indicated that recombinant (r) SV-66 but not rSV-170 inhibited plasma coagulation.Functional assays indicated that rSV66 reduced the activity of ten serine proteases, including several involved in mammalian coagulation. rSV-66 most strongly inhibited the activity of Factor Xa, elastase, and cathepsin G, exhibited lesser inhibitory activity against Factor IXa, Factor XIa, and plasmin, and exhibited no activity against Factor XIIa and thrombin.We conclude that Simukunin preferentially inhibits Factor Xa.

View Article: PubMed Central - PubMed

Affiliation: Department of Entomology, The University of Georgia, Athens, Georgia, United States of America.

ABSTRACT

Background: Black flies (Diptera: Simuliidae) feed on blood, and are important vectors of Onchocerca volvulus, the etiolytic agent of River Blindness. Blood feeding depends on pharmacological properties of saliva, including anticoagulation, but the molecules responsible for this activity have not been well characterized.

Methodology/principal findings: Two Kunitz family proteins, SV-66 and SV-170, were identified in the sialome of the black fly Simulium vittatum. As Kunitz proteins are inhibitors of serine proteases, we hypothesized that SV-66 and/or -170 were involved in the anticoagulant activity of black fly saliva. Our results indicated that recombinant (r) SV-66 but not rSV-170 inhibited plasma coagulation. Mutational analysis suggested that SV-66 is a canonical BPTI-like inhibitor. Functional assays indicated that rSV66 reduced the activity of ten serine proteases, including several involved in mammalian coagulation. rSV-66 most strongly inhibited the activity of Factor Xa, elastase, and cathepsin G, exhibited lesser inhibitory activity against Factor IXa, Factor XIa, and plasmin, and exhibited no activity against Factor XIIa and thrombin. Surface plasmon resonance studies indicated that rSV-66 bound with highest affinity to elastase (K(D) = 0.4 nM) and to the active site of FXa (K(D) = 3.07 nM). We propose the name "Simukunin" for this novel protein.

Conclusions: We conclude that Simukunin preferentially inhibits Factor Xa. The inhibition of elastase and cathepsin G further suggests this protein may modulate inflammation, which could potentially affect pathogen transmission.

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Related in: MedlinePlus

rSimukunin displays high-affinity binding to FXa and elastase.(A) Surface Plasmon Resonance (SPR) Sensorgrams show mouse, bovine, and human FXa, the FXa derivatives des-GLA-hFXa and DEGR-hFXa, and other coagulation factors (all tested at 200 nM) binding to immobilized rSimukunin. (B) Sensorgrams for various concentrations of human FXa (in nM: purple, 50; red, 25; orange, 12.5; green, 6.25, blue, 3.1) binding to immobilized rSumukunin. (C) Sensorgrams for various concentrations of elastase (in nM: red, 3.75; orange, 1.8; green, 0.9; blue, 0.45) binding to immobilized rSimukunin. Data were fitted using a 1∶1 binding model (Langmuir). RU: resonance units.
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pone-0029964-g006: rSimukunin displays high-affinity binding to FXa and elastase.(A) Surface Plasmon Resonance (SPR) Sensorgrams show mouse, bovine, and human FXa, the FXa derivatives des-GLA-hFXa and DEGR-hFXa, and other coagulation factors (all tested at 200 nM) binding to immobilized rSimukunin. (B) Sensorgrams for various concentrations of human FXa (in nM: purple, 50; red, 25; orange, 12.5; green, 6.25, blue, 3.1) binding to immobilized rSumukunin. (C) Sensorgrams for various concentrations of elastase (in nM: red, 3.75; orange, 1.8; green, 0.9; blue, 0.45) binding to immobilized rSimukunin. Data were fitted using a 1∶1 binding model (Langmuir). RU: resonance units.

Mentions: The preceding results provided measures of affinity for elastase, FXa, and cathepsin G by rSimukinun, but were insufficient for calculation of binding and dissociation kinetics. We therefore conducted Surface Plasmon Resonance (SPR) studies using rSimukunin immobilized on a sensor chip and used selected target enzymes as the analyte. These studies showed that rSimukunin bound FXa from several mammals (human, mouse and bovine) with high affinity (Figure 6A). rSimukunin also bound DES-Gla-FXa, an FXa derivative lacking the Gla domain necessary for docking onto a negatively charged membrane surface. In contrast, rSimukunin did not bind DEGR-FXa, a derivative blocked at the active site, or FX, which is the zymogen precursor of FXa (Figure 6A). rSimukunin also exhibited very weak binding responses to FIXa and FXIa, and no binding responses to FVIIa, FXIIIa or thrombin (Figure 6A). Kinetic analysis determined that FXa bound rSimukunin with a KD of 3.07 nM (Figure 6B and Table 2), but assays with elastase revealed an even stronger affinity for rSimukunin with a KD = ∼0.4 nM (Figure 6C and Table 2). These KD values are consistent with the Ki values we calculated based on the Km and IC50. The low KD values for both FXa and elastase result from a fast association rate (ka1 = 4.3×107 M−1 s−1 for elastase) and a very slow off rate (kd1 = 0.017 s−1) indicative of rSimukunin functioning as a tight-binding inhibitor.


Simukunin from the salivary glands of the black fly Simulium vittatum inhibits enzymes that regulate clotting and inflammatory responses.

Tsujimoto H, Kotsyfakis M, Francischetti IM, Eum JH, Strand MR, Champagne DE - PLoS ONE (2012)

rSimukunin displays high-affinity binding to FXa and elastase.(A) Surface Plasmon Resonance (SPR) Sensorgrams show mouse, bovine, and human FXa, the FXa derivatives des-GLA-hFXa and DEGR-hFXa, and other coagulation factors (all tested at 200 nM) binding to immobilized rSimukunin. (B) Sensorgrams for various concentrations of human FXa (in nM: purple, 50; red, 25; orange, 12.5; green, 6.25, blue, 3.1) binding to immobilized rSumukunin. (C) Sensorgrams for various concentrations of elastase (in nM: red, 3.75; orange, 1.8; green, 0.9; blue, 0.45) binding to immobilized rSimukunin. Data were fitted using a 1∶1 binding model (Langmuir). RU: resonance units.
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pone-0029964-g006: rSimukunin displays high-affinity binding to FXa and elastase.(A) Surface Plasmon Resonance (SPR) Sensorgrams show mouse, bovine, and human FXa, the FXa derivatives des-GLA-hFXa and DEGR-hFXa, and other coagulation factors (all tested at 200 nM) binding to immobilized rSimukunin. (B) Sensorgrams for various concentrations of human FXa (in nM: purple, 50; red, 25; orange, 12.5; green, 6.25, blue, 3.1) binding to immobilized rSumukunin. (C) Sensorgrams for various concentrations of elastase (in nM: red, 3.75; orange, 1.8; green, 0.9; blue, 0.45) binding to immobilized rSimukunin. Data were fitted using a 1∶1 binding model (Langmuir). RU: resonance units.
Mentions: The preceding results provided measures of affinity for elastase, FXa, and cathepsin G by rSimukinun, but were insufficient for calculation of binding and dissociation kinetics. We therefore conducted Surface Plasmon Resonance (SPR) studies using rSimukunin immobilized on a sensor chip and used selected target enzymes as the analyte. These studies showed that rSimukunin bound FXa from several mammals (human, mouse and bovine) with high affinity (Figure 6A). rSimukunin also bound DES-Gla-FXa, an FXa derivative lacking the Gla domain necessary for docking onto a negatively charged membrane surface. In contrast, rSimukunin did not bind DEGR-FXa, a derivative blocked at the active site, or FX, which is the zymogen precursor of FXa (Figure 6A). rSimukunin also exhibited very weak binding responses to FIXa and FXIa, and no binding responses to FVIIa, FXIIIa or thrombin (Figure 6A). Kinetic analysis determined that FXa bound rSimukunin with a KD of 3.07 nM (Figure 6B and Table 2), but assays with elastase revealed an even stronger affinity for rSimukunin with a KD = ∼0.4 nM (Figure 6C and Table 2). These KD values are consistent with the Ki values we calculated based on the Km and IC50. The low KD values for both FXa and elastase result from a fast association rate (ka1 = 4.3×107 M−1 s−1 for elastase) and a very slow off rate (kd1 = 0.017 s−1) indicative of rSimukunin functioning as a tight-binding inhibitor.

Bottom Line: Our results indicated that recombinant (r) SV-66 but not rSV-170 inhibited plasma coagulation.Functional assays indicated that rSV66 reduced the activity of ten serine proteases, including several involved in mammalian coagulation. rSV-66 most strongly inhibited the activity of Factor Xa, elastase, and cathepsin G, exhibited lesser inhibitory activity against Factor IXa, Factor XIa, and plasmin, and exhibited no activity against Factor XIIa and thrombin.We conclude that Simukunin preferentially inhibits Factor Xa.

View Article: PubMed Central - PubMed

Affiliation: Department of Entomology, The University of Georgia, Athens, Georgia, United States of America.

ABSTRACT

Background: Black flies (Diptera: Simuliidae) feed on blood, and are important vectors of Onchocerca volvulus, the etiolytic agent of River Blindness. Blood feeding depends on pharmacological properties of saliva, including anticoagulation, but the molecules responsible for this activity have not been well characterized.

Methodology/principal findings: Two Kunitz family proteins, SV-66 and SV-170, were identified in the sialome of the black fly Simulium vittatum. As Kunitz proteins are inhibitors of serine proteases, we hypothesized that SV-66 and/or -170 were involved in the anticoagulant activity of black fly saliva. Our results indicated that recombinant (r) SV-66 but not rSV-170 inhibited plasma coagulation. Mutational analysis suggested that SV-66 is a canonical BPTI-like inhibitor. Functional assays indicated that rSV66 reduced the activity of ten serine proteases, including several involved in mammalian coagulation. rSV-66 most strongly inhibited the activity of Factor Xa, elastase, and cathepsin G, exhibited lesser inhibitory activity against Factor IXa, Factor XIa, and plasmin, and exhibited no activity against Factor XIIa and thrombin. Surface plasmon resonance studies indicated that rSV-66 bound with highest affinity to elastase (K(D) = 0.4 nM) and to the active site of FXa (K(D) = 3.07 nM). We propose the name "Simukunin" for this novel protein.

Conclusions: We conclude that Simukunin preferentially inhibits Factor Xa. The inhibition of elastase and cathepsin G further suggests this protein may modulate inflammation, which could potentially affect pathogen transmission.

Show MeSH
Related in: MedlinePlus