Abstract
BackgroundBlack 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 FindingsTwo 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 (KD = 0.4 nM) and to the active site of FXa (KD = 3.07 nM). We propose the name “Simukunin” for this novel protein.ConclusionsWe 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.
Highlights
Both eukaryotes and prokaryotes produce Kunitz family protease inhibitors, which indicates an ancient origin for Kunitz family encoding genes [1,2]
We conclude that Simukunin preferentially inhibits Factor Xa
Our results indicated that SV-66 is an anti-coagulant with anti-FXa activity that inhibits several other serine proteases
Summary
Both eukaryotes and prokaryotes produce Kunitz family protease inhibitors, which indicates an ancient origin for Kunitz family encoding genes [1,2]. The most conserved function of Kunitz family proteins is the reversible competitive inhibition of serine proteases [1]. A single Kunitz domain is small (,60 aa) and forms a compact globular fold typically containing three disulfide bonds. Based on the structure of bovine pancreatic trypsin inhibitor (BPTI), a typical Kunitz domain contains cysteine residues at positions 5, 14, 30, 38, 51 and 55 in the mature peptide, which form three disulfide bonds C5–C55, C14–C38 and C30–C51 [1]. Many Kunitz domains act as protease inhibitors through their scissile bond at positions 15 (P1) and 16 (P19). 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
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