Fig 1.
Titration assays of recombinant EgKUs: results for EgKU-3 and EgKU-4.
Increasing concentrations of bovine chymotrypsin or trypsin were pre-incubated with fixed amounts of recombinant EgKU-3 (A) or EgKU-4 (B), respectively, and mixed with the corresponding enzyme substrate. The plots show the initial steady-state rate of substrate hydrolysis for each enzyme concentration; the activity in the absence of inhibitor is indicated in grey. (A) EgKU-3 is a high affinity inhibitor of chymotrypsin. Note that the slope at the enzyme concentrations for which activity is detected compares very well with the slope in the absence of inhibitor. The x-intercept of this plot (1.5 nM) represents the enzyme concentration interacting with 1.5 nM of EgKU-3. Thus, EgKU-3 inhibits chymotrypsin with a 1:1 stoichiometry. (B) EgKU-4 is a low affinity inhibitor of trypsin. Note that trypsin activity is detected all over the assayed enzyme range in the presence of an inhibitor concentration 1000-fold higher than the peptidase concentration. Representative results are shown. Experiments with EgKU-3 and EgKU-4 were carried out five and two independent times, respectively. Within each experiment, measurements were performed in duplicates.
Table 1.
Screening of serine peptidase inhibitory activity of EgKUs.
Fig 2.
Inhibition studies with EgKU-3: results for bovine chymotrypsin A.
(A) Enzyme inhibition. The enzyme (1 nM) was preincubated for 15 min with EgKU-3 (0.1–3.0 nM) and mixed with substrate (Suc-Ala-Ala-Pro-Phe-AMC, 5 μM) in 50 mM Tris-HCl, pH 8.0, 0.01% Triton X-100, at 37°C. Initial steady-state rate measurements were performed in duplicates and the experiment was repeated 5 independent times. A representative experiment is shown. KI*app values at equilibrium were determined using Eq (1) for tight binding inhibitors as described in Materials and Methods. The solid line represents the best fit to this equation. (B) Representative progress curves for the inhibition. The enzyme (1 nM) was added to reaction mixtures containing the substrate (Suc-Ala-Ala-Pro-Phe-AMC, 5 μM) and increasing concentrations of EgKU-3 (0, 0.5, 1, 2, 3, 6, and 15 nM, gray traces) in 50 mM Tris-HCl, pH 8.0, 0.01% Triton X-100, at 37°C. The black traces represent the best fit to Eq 3, from which kobs were obtained. (C) Dependence of kobs on the concentration of inhibitor. The enzyme was added to reaction mixtures containing the substrate (Suc-Ala-Ala-Pro-Phe-AMC, 5 μM) and increasing concentration of EgKU-3 in 50 mM Tris-HCl, pH 8.0, 0.01% Triton X-100, at 37°C. The enzyme concentrations were: 1 nM for 0.5–3 nM of EgKU-3, 2 nM for 3–6 nM of EgKU-3, and 3 nM for 6–10 nM of EgKU-3. kobs values were obtained from time course experiments according to Eq 3 and correspond to the average of at least two time courses. The black trace represents the best fit to Eq 5 in agreement with Eq 4. The experiment was repeated 3 independent times.
Table 2.
Global inhibition constants (KI*) of EgKU-3, EgKU-4 and EgKU-8 acting on pancreatic serine peptidases.
Table 3.
Inhibitory kinetics of EgKU-3 on bovine chymotrypsin A.
Fig 3.
Inhibition studies with EgKU-1 and EgKU-4: results for Kv from DRG neurons.
Representative experiments showing that recombinant EgKU-1 (200 nM) (A) blocks voltage dependent K+ currents elicited by a pulse of -100 to 0 mV during 800 ms (holding potential Vh = -60 mV); and (B) that the inhibition effect is only partially reversible after washout of the inhibitor. (C)–(F) Effect of the EgKUs on K+ currents activated by increasing voltage pulses. The K+ currents were recorded following stepwise increments of 10 mV of the membrane voltage between -110 and 30 mV from a holding potential of -60 mV. Recordings showing the effect of recombinant EgKU-1 (200 nM) are shown in (C) and the current-voltage relationship of these traces in (D). Similar analyses with native EgKU-1 (100 nM) and recombinant EgKU-4 (200 nM) are shown in (E) and (F), respectively. The black traces correspond to control conditions and the gray ones after EgKU perfusion. Note that the effects of native and recombinant EgKU-1 are of the same order.
Fig 4.
Studies with EgKU-3 and EgKU-8 on total K+ currents from DRG neurons.
Representative experiments showing that recombinant EgKU-3 and EgKU-8 (1 μM) do not block voltage-dependent K+ currents elicited by a pulse of -100 to 0 mV during 800 ms (Vh = -60 mV). The superimposed traces correspond to control recordings (black) and records after the perfusion of each EgKU (red). Positive and negative controls were carried out in parallel, using α-DTX (100 nM) and albumin (15 μM), respectively.
Fig 5.
Inhibition studies with EgKU-1 on isolated K+ currents from DRG neurons.
Blocking effect of recombinant EgKU-1 (675 nM) on isolated K+ currents activated by increasing voltage pulses. (A) Voltage-dependent K+ currents (fast -transient A-type- currents, IKA; as well as non inactivating -delayed-rectifier- currents, IKDR) were recorded from a holding potential of -100 mV, following stepwise increments of 10 mV of the membrane voltage, between -65 and 55 mV. (B) IKDR currents were similarly recorded from a holding potential of -45mV, so as to inactivate IKA currents. (C) and (D) are the corresponding current-voltage plots of (A) and (B), whereas (E) is the current-voltage plot accounting for IKA currents and was obtained by subtracting (D) from (C).
Fig 6.
Concentration-response analysis of native EgKU-1 on total K+ currents from DRG neurons.
(A) Representative traces showing total K+ currents elicited by a voltage pulse of -100 to 0 mV during 1000 ms (as indicated above the current trace) under control conditions, after 1 min perfusion of 200 nM of native EgKU-1 and after washing. (B) Concentration-response analysis of EgKU-1 inhibitory effect on K+ currents, measured at the end of the voltage pulse, on the steady-state component of the current. The black line shows the best fit to the dose-response equation, from which the IC50 was calculated (216 ± 26 nM). The data correspond to the mean ± standard error (n = 5 in all cases). The asterisks indicate Student’s t-test significance with respect to the effect in the absence of inhibitor (P ≤ 0.05).
Fig 7.
Inhibition studies with EgKU-1 and EgKU-4: results for ASIC currents from DRG neurons.
(A-C) Representative traces showing the acid (pH 6.1, 5 s) activated current under control conditions (left), after sustained (25 s) perfusion of 30 nM of each EgKU (center) and after 1 min washout of the inhibitors (right). Note that EgKU-1 and EgKU-4 reduced the amplitude of the Na+ current, that recombinant EgKU-1 reproduced the effect of the native inhibitor and that the recovery after washout was higher than 90% in all cases. (D-E) Representative traces from analogous assays with 30 nM of EgKU-3 and EgKU-8. The slight decrement of the current amplitude induced by EgKU-3 was significant (see the text for further details); EgKU-8 had no effect. (F) Albumin (15 μM) was used as negative control. Calibration in each case applies to the control, effect and washout recordings of each panel.
Fig 8.
Concentration-response analysis of native EgKU-1 on ASIC currents from DRG neurons.
(A) Analysis of native EgKU-1 inhibitory effect on the ASIC current amplitude (n = 26). The black line shows the best fit to the dose-response equation, from which the IC50 was calculated (7.8 ± 0.7 nM). The data correspond to the mean ± standard error (n ≥ 6 in all cases, except for 1 nM in which n = 4). The asterisks indicate Student’s t-test significance with respect to the effect in the absence of inhibitor (P ≤ 0.05). (B) and (C) correspond to positive and negative controls, respectively. (B) Representative traces showing the acid (pH 6.1, 5 s) activated current under control conditions (left), after sustained (25 s) perfusion of α-DTX (center), and after 1 min washout (right). α-DTX (1 μM; n = 6) significantly decreased the current amplitude (44.5 ± 7.0%; P = 0.045). (C) The application of EgKU-1 in extracellular solution, without any pH change, had no effect.
Fig 9.
Detection of EgKU-3 and EgKU-8 in parasite secretions.
Analysis by MALDI-TOF MS of hydatid fluid from a bovine cyst (A), as well as of chymotrypsin-affinity purified fractions from the same sample (B) and from the supernatant of cultured immature adults (C). Signals whose m/z values could derive from the EgKUs are indicated (MH+ predicted for mature EgKU-3 and EgKU-8 are: 6406.8 and 6520.9, respectively). Note that the signals putatively corresponding to the EgKUs are significantly enriched in the eluate from the affinity matrix. The identity of EgKU-3 and EgKU-8 purified from cyst fluid was subsequently confirmed by peptide mass fingerprinting (see S1 Dataset and the text for further details), as previously described [3].
Fig 10.
Structural analyses of EgKU-1/EgKU-4 and EgKU-3/EgKU-8.
(A) Cartoon representation of structural models from the EgKUs and the crystal structure of α-DTX (1DTX) featuring solvent-accessible (> 40 Å2) aromatic (purple), acid (red) and basic (blue) residues. N and C terminal ends are labeled. Note the presence of patches of basic amino acids with close aromatic residues in the models of α-DTX, EgKU-1 and EgKU-4. (B) Molecular surface electrostatic representations of the same proteins in the same orientation, highlighting global differences in charge distribution; scale represents charge from positive blue to negative red. (C) Sequence alignment produced with TEXshade [82] and hand-edited, featuring aromatic, acid and basic residues; those with solvent-accessibilities < 40 Å2 are grey shaded. Note that structurally equivalent positions in the EgKUs and α-DTX are shifted two residues in the primary sequence. The P1 site of serine peptidase inhibitors, located at the center of the antipeptidase loop, is indicated with arrowheads in (A) and (C).
Fig 11.
Expanded view of the E. granulosus Kunitz family.
Unrooted phylogenetic tree highlighting sequence groupings within the family that roughly correlate with functional features described in the main text. Of outmost notice are sub-clades which include pairs such as the serine peptidase inhibitors EgKU-3/EgKU-8 (red clade) and EgKU-6/EgKU-7 (green clade); and the channel blockers EgKU-1/EgKU-4 (blue clade). Note that the sequences from T. solium pair with their close E. granulosus paralogs. Interestingly, the serine peptidase inhibitors SjKI-1 [37], SmKI-1 [38] and EGR_07242 (EgKI-2 in [36]) group in the red clade. The sequences from F. hepatica (FhKTM [42] and FhKT1 [43]) define a basal, separate clade that could reflect functional diversity (cysteine peptidase inhibition; [43]). The long branch of EgKU-2 (and its putative T. solium ortholog) may reflect either a basal position of the protein (ancient/extreme sequence divergence), an accelerated evolution (e.g. through positive selection) or even relaxed selective pressures resulting in high tolerance to mutation accumulation. Data are insufficient to distinguish between such alternative scenarios. EgrG_001136500, in a black clade to the left, was also found to be a potent serine peptidase inhibitor (EgKI-1 in [36]). The position of the mollusk sequence (Conkunitzin S1), which was characterized as a channel blocker [14], is probably derived from the fact that, similar to EGR_07242, it lacks Cys14. This artifact is to be expected in short sequences. Bottom scale bar denotes average substitutions per site.