Simukunin from the Salivary Glands of the Black Fly Simulium vittatum Inhibits Enzymes That Regulate Clotting and Inflammatory Responses

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

SV-66 is constitutively expressed in the salivary glands of adult female <i>S. vittatum</i>.

<p>(A) Sex and tissue-specific expression of Simukunin. Transcript was detected in the adult female body (head and thorax without abdomen), but not in adult female carcasses (bodies without salivary glands or heads). M: male; F: mature female; NTC: no-template control. 5 individuals were pooled for each sample. Actin PCR products are shown as a positive control indicating equivalent concentrations of template among samples. Each panel is a composite of two rows (upper and lower) of wells, run in the same gel at the same time. (B) Time-course of expression of Simukunin. Transcript was detected before, and at selected time points up to 48 h post blood meal. Fresh: freshly eclosed non-blood-fed female adult; hpbf: hours post blood feeding; NTC: no-template control. Two samples, each comprised of 5 pooled individuals, were analyzed for each time point.</p

SV-66 and SV-170 belong to the Kunitz family of protease inhibitors.

<p>(A) Nucleotide and translated polypeptide sequences of SV-66 and SV-170. Start and stop codons are in white with black shading. Numbers below the amino acid residues are designated based on the putative mature protein. Signal sequences predicted by SignalP are underlined. Top: SV-66 encodes a 102 amino-acid polypeptide (Simukunin), which includes a 19 amino-acid N-terminal signal sequence. Mature Simukunin is predicted to consist of 83 amino-acid residues, with a theoretical mass of 9627.22 Da and pI of 9.93. SV-66 also contains a putative O-glycosylation site at position 81 (Ser). Bottom: SV-170 encodes a 78 amino-acid polypeptide, which includes an N-terminal 22 amino-acid signal sequence. Mature SV-170 is predicted to consist of 56 amino-acid residues, and theoretical mass and pI are 6526.66 Da and 8.87, respectively. (B) Alignment of representative Kunitz domain sequences with SV-66 and SV-170. Each Kunitz domain was separated from the original sequences for alignment (numbers denote amino-acid positions in the original mature peptides). All reference sequences were retrieved from GenBank. Accession numbers are: TFPI (human: 3 Kunitz domains), P10646; BPTI (<i>Bos taurus</i>: 1 Kunitz domain), AAI49369; Amblin (<i>Amblyomma hebraeum</i>: 2 Kunitz domains), AAR97367; Boophilin (<i>Rhipicephalus microplus</i>: 2 Kunitz domains), CAC82583. Strictly conserved cysteine residues are white with green shading, and predicted conserved disulfide bonds are shown in solid lines. The reactive site loop (RSL) P₄-P₂′ residues, conserved in canonical binding inhibitors, are indicated by asterisks. The P₁ residue is indicated with an arrow. Highly conserved P₁-P₁′ (Arg/Lys-Ala/Gly) residues are shown in white with purple shading. Other identical residues across the domain are shaded with yellow and conserved or semi-conserved residues are shaded with grey.</p

Kinetics of FXa and Elastase interactions with rSimukunin.

<p>Responses were obtained by injecting FXa or elastase over immobilized rSimukunin for 180 seconds at a flow rate of 30 µl/minute. Data were derived from <i>Ka1</i> and <i>Kd1</i> and fitted using the Langmuir (1∶1 binding) equation. Kinetic values obtained from the sensorgrams presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029964#pone-0029964-g006" target="_blank">Figure 6B and 6C</a> for FXa and elastase respectively.</p

rSimukunin displays high-affinity binding to FXa and elastase.

<p>(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.</p

rSV-66 delays clotting of human plasma.

<p>Indicated concentrations of rSimukunin (rSV-66) and rSV-170 were tested by the recalcification time assay. Citrated human plasma (50 µl) was mixed with recombinant proteins (in 50 µl 0.15 M NaCl, 10 mM HEPES pH7.4) and pre-warmed at 37°C for 15 min before clotting was initiated by the addition of 50 µl prewarmed CaCl₂ (25 mM). Recalcification (clotting) time was determined by monitoring absorbance at 650 nm at 10-sec intervals in a SpectraMax 340 microtiter plate reader, with onset time (the time to a linear increase in the OD, which reflects the maximal rate of formation of insoluble fibrin) set at an OD of 0.04. Clotting times (mean ± SD) for rSimukunin and rSV-170 are shown in black bars and grey bars, respectively. The white bar is the Ca²⁺-only control. One-way analysis of variance indicted a significant difference between treatments (F_{6, 21} = 119.3; P<0.001) for rSimukunin but not rSV-170. Subsequent multiple comparisons between various treatments and the positive control were performed using the Holm-Sidak method. Statistically significant increases in clotting time at <i>p</i><0.05 and <i>p</i><0.01 are indicated by * and ** respectively. Results shown are representative of three independent experiments.</p

IC₅₀ values for rSimukunin against various serine proteases.

<p>Titrated concentrations of rSimukunin were tested with constant concentrations of enzymes (in Concentration column) to determine the concentrations of rSimukunin that gave a 50% inhibition of the enzyme activity. Ratios of IC₅₀ to enzyme concentration are also shown as different concentrations of enzymes were necessary to obtain linear reaction rates. Titration curves are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029964#pone.0029964.s002" target="_blank">Figure S2</a>.</p

Point mutations in the reactive site loop of rSimulkunin disable anti-coagulation activity.

<p>Each recombinant protein was tested by adding 0.5 µg (grey bars) or 1 µg (black bars) to a fixed volume of plasma. Plasma was then pre-warmed at 37°C for 15 min before addition of 25 mM (8.3 mM final concentration) CaCl₂ (pre-warmed) to initiate clotting. White bar shows the Ca²⁺-only plasma control. The graph shows mean ± SD from three independently conducted experiments. <i>p</i>-values for significant differences by one-tailed t-test are shown, where the alternative hypothesis is that sample recalcification time is greater than the Ca²⁺-only control. Statistically significant increases in clotting time at <i>p</i><0.05 and <i>p</i><0.01 are indicated by * and ** respectively.</p

Ovary ecdysteroidogenic hormone functions independently of the insulin receptor in the yellow fever mosquito, Aedes aegypti

Most mosquito species must feed on the blood of a vertebrate host to produce eggs. In the yellow fever mosquito, Aedes aegypti, blood feeding triggers medial neurosecretory cells in the brain to release insulin-like peptides (ILPs) and ovary ecdysteroidogenic hormone (OEH). Theses hormones thereafter directly induce the ovaries to produce ecdysteroid hormone (ECD), which activates the synthesis of yolk proteins in the fat body for uptake by oocytes. ILP3 stimulates ECD production by binding to the mosquito insulin receptor (MIR). In contrast, little is known about the mode of action of OEH, which is a member of a neuropeptide family called neuroparsin. Here we report that OEH is the only neuroparsin family member present in the Ae. aegypti genome and that other mosquitoes also encode only one neuroparsin gene. Immunoblotting experiments suggested that the full-length form of the peptide, which we call long OEH (lOEH), is processed into short OEH (sOEH). The importance of processing, however, remained unclear because a recombinant form of lOEH (rlOEH) and synthetic sOEH exhibited very similar biological activity. A series of experiments indicated that neither rlOEH nor sOEH bound to ILP3 or the MIR. Signaling studies further showed that ILP3 activated the MIR but rlOEH did not, yet both neuropeptides activated Akt, which is a marker for insulin pathway signaling. Our results also indicated that activation of TOR signaling in the ovaries required co-stimulation by amino acids and either ILP3 or rlOEH. Overall, we conclude that OEH activates the insulin signaling pathway independently of the MIR, and that insulin and TOR signaling in the ovaries is coupled