Amblyomma americanum tick saliva serine protease inhibitor 6 is a cross-class inhibitor of serine proteases and papain-like cysteine proteases that delays plasma clotting and inhibits platelet aggregation

Abstract

We previously demonstrated that Amblyomma americanum tick serine protease inhibitor 6 (AamS6) was secreted into the host during tick feeding and that both its mRNA and protein were ubiquitously and highly expressed during the first 3 days of tick feeding. This study demonstrates that AamS6 is a cross-class inhibitor of both serine- and papain-like cysteine proteases that has apparent antihaemostatic functions. Consistent with the typical inhibitory serpin characteristics, enzyme kinetics analyses revealed that Pichia pastoris-expressed recombinant (r) AamS6 reduced initial velocities of substrate hydrolysis (V₀) and/or maximum enzyme velocity (V(max)) of trypsin, chymotrypsin, elastase, chymase, and papain in a dose-response manner. We speculate that rAamS6 inhibited plasmin in a temporary fashion in that while rAamS6 reduced V₀ of plasmin by up to ∼53%, it had no effect on V(max). Our data also suggest that rAmS6 has minimal or no apparent effect on V₀ or V(max) of thrombin, factor Xa, and kallikrein. We speculate that AamS6 is apparently involved in facilitating blood meal feeding in that various amounts of rAamS6 reduced platelet aggregation by up to ∼47% and delayed plasma clotting time in the recalcification time assay by up to ∼210 s. AamS6 is most likely not involved with the tick's evasion of the host's complement defense mechanism, in that rAamS6 did not interfere with the complement activation pathway. Findings in this study are discussed in the context of expanding our understanding of tick proteins that control bloodmeal feeding and hence tick-borne disease transmission by ticks.

Figure 1

Expression and affinity purification of recombinant (r) Amblyomma americanum tick saliva serine protease inhibitor (AamS6) in Pichia pastoris. The mature AamS6 protein cDNA was cloned into the pPICZα plasmid and elctroporated into X33 P. pastoris strain as described. Positive transformants were selected for methanol utilization on yeast agar plates. Selected colonies were grown in culture to A₆₀₀ of 1 before inducing rAamS6 expression by daily feeding of cultures with methanol to 5% final concentration. Panel A = daily (lanes 1–5) rAamS6 expression detection by Western blotting using the antibody to c-terminus histidine tag. Panel B = Affinity purified rAamS6 electrophored on a 4–16% gradient native acrylamide gel and silver stained. CW = column wash, EP = column eluted protein. Panel C = Validation of N-glycosylation posttranslational modification of rAamS6. + DE = rAamS6 treated with the deglycosylating PngaseF enzyme, −DE = non-treated rAamS6.

Figure 2

Protease inhibitor function profiling of rAamS6. Indicated candidate proteases (500 ng) were co-incubated with affinity purified, 0.34 μg and 1.4 μg or 0.066 μM and 0.27 μM for 15 min at 25 °C. After incubation, appropriate peptide substrates were added to final concentrations indicated in materials and methods. Subsequently the release of the chromophore, p-nitroanilide as proxy for substrate hydrolysis was monitored at A₄₁₀ using the VersaMax microplate reader. The observed A₄₁₀ were converted to released μM concentrations of digested peptides as described in materials and methods. Non-linear regression in Graphpad software was used to fit the second order polynomial and the Michaelis and Menten equations on our data to respectively estimate (A) the initial velocities of substrate hydrolysis, and (B) the maximum velocity of substrate hydrolysis.

Figure 2

Protease inhibitor function profiling of rAamS6. Indicated candidate proteases (500 ng) were co-incubated with affinity purified, 0.34 μg and 1.4 μg or 0.066 μM and 0.27 μM for 15 min at 25 °C. After incubation, appropriate peptide substrates were added to final concentrations indicated in materials and methods. Subsequently the release of the chromophore, p-nitroanilide as proxy for substrate hydrolysis was monitored at A₄₁₀ using the VersaMax microplate reader. The observed A₄₁₀ were converted to released μM concentrations of digested peptides as described in materials and methods. Non-linear regression in Graphpad software was used to fit the second order polynomial and the Michaelis and Menten equations on our data to respectively estimate (A) the initial velocities of substrate hydrolysis, and (B) the maximum velocity of substrate hydrolysis.

Figure 3

Effect of recombinant Amblyomma americanum tick saliva serine protease inhibitor (rAamS6) on platelet aggregation: Citrated cattle whole diluted 1:1 with normal saline was co-incubated without (A) or with (B and C) rAamS6, 2 and 12 μg or 0.08 and 0.27 μM at 37 °C for 10 min. Subsequently platelet aggregation was induced by adding adenosine diphosphate to final 20 μM concentration. Platelet aggregation was monitored using the whole blood platelet aggregometer as described under materials and methods.

Figure 4

Effect of affinity purified on plasma clotting time: Citrated human plasma depleted of platelets was co-incubated with various amounts (0.48, 0.40, 0.32, 0.24, 0.16 and 0.08 μg or 0.13, 0.11, 0.088, 0.066, 0.044 and 0.022 μM) of rAamS6 at 37 °C for 10 min. Adding 25 mM calcium chloride triggered plasma clotting. Clot formation was monitored at A₆₅₀ using the VersaMax microplate reader. The solid line arrowhead at A₆₅₀ indicates plasma clotting starting point. The broken line arrowheads, A, B, C, D, E, F and G mark the plasma clotting start times at 180, 190, 220, 250, 270, 310 and 390 s when plasma was co-incubated with 0, 0.022, 0.044, 0.066, 0.088, 0.11 and 0.13 μM affinity purified rAamS6, respectively.