Fig 1.
AAS27 is highly transcribed at adult unfed stage.
(A-C) AAS27 relative expression analysis in unfed (UN), 24, 48, 72, 96, and 120 h fed adult female tick dissected tissues: salivary glands (SG), midguts (MG) and remnants as carcass (CA) analyzed by qRT-PCR. In three biological replicates, relative expression levels of AAS27 were analyzed using the 2−ΔΔCt method using ribosomal protein S4 (RPS4) transcript as internal reference. The lowest expressed time point was used as a calibrator for each tissue. (D-G) AAS27 qualitative expression analysis were performed by western blot in in unfed (UN), 24, 72, and 120h fed tick dissected salivary glands (SG), midguts (MG), synganglion (SYN), Malpighian tubules (MT), ovary (OV) and remnants as carcass (CA), eggs 22 days after oviposition (E), unfed larvae (L), unfed nymphs (N), unfed adult females (F) and males (M). Protein extracts were subjected to western blotting analyses using a monospecific antibody (purified) against yeast-expressed rAAS27 (0.30 μg/μL in a 1:500 dilution). rAASrAAS-27 (500 ng) was used as positive control (C+).
Fig 2.
rAAS27 inhibits trypsin-like serine proteases.
rAAS27 (1 μM) was tested against 17 different host-derived serine proteases, including: rat trypin IV (20 nM), pancreatic bovine α-chymotrypsin (1.4 nM), pancreatic porcine elastase (61.8 nM), human proteinase-3 (280 nM), human chymase (21.7 nM), pancreatic bovine trypsin (0.3 nM), pancreatic porcine kallikrein (10 nM), human thrombin (19.2 nM), human neutrophil cathepsin G (425.5 nM), human plasmin (5.3 nM), human factor XIa (3.7 nM), bovine factor IXa (314.4 nM), human factor XIIa (15 nM), human neutrophil elastase (14.9 nM), human t-PA (23.6 nM), human u-PA (29.6 nM), and human factor Xa (2.3 nM). rAAS27 and protease were pre-incubated for 15 minutes at 37°C in 20 mM Tris-HCl, 150 mM NaCl, BSA 0.1%, pH 7.4 buffer. The corresponding substrate for each enzyme was added in a 100 μL final reaction volume and substrate hydrolysis was measured at OD405nm every 11s for 15 min at 30°C. The percent enzyme activity inhibition level was determined as described in Material and Methods section. Data are presented as mean ± standard deviation of three independent replicate readings.
Fig 3.
rAAS27 is an effective inhibitor of trypsin and plasmin.
Residual protease activity in the presence and absence of rAAS27 was evaluated by pre-incubating rAAS27 for 1 h at 37°C with (A) trypsin (2 nM), (B) plasmin (34 nM), (C) chymotrypsin (2.8 nM), and (D) factor XIa (3.7 nM), resulting in molar ratios (serpin:protease) ranging from 0 to 10. Protease activity was measured using specific colorimetric substrate for each protease as described in Materials and Methods. The data were plotted as residual protease activity (Vi/V0) versus molar ratio (serpin:protease). The SI was determined by linear regression to the initial points of inhibitory curve. Data are presented as mean ± standard deviation of three independent replicate readings.
Fig 4.
rAAS27 forms heat and SDS-stable complex with trypsin and plasmin.
Increasing amounts of rAAS27 were pre-incubated for 1 h at 37°C with a constant concentration of (A) trypsin (0.1 μg) and (B) plasmin (0.2 μg), resulting in molar ratios varying from 0.6:1 to 10:1 (serpin:protease). Samples were resolved on 12% SDS–PAGE and silver-stained to identify SDS-stable complexes (indicated by arrows).
Fig 5.
rAAS27 is an effective inhibitor of trypsin.
Discontinuous assay of the inhibition of trypsin by rAAS27. (A) Semilogarithmic plots of residual enzymatic activity of trypsin versus time of incubation for reactions at various concentrations of rAAS27 (2.5–25 nM). (B) Plot of kobs as a function of rAAS27 concentration. Linear regression of the slope represents the second-order rate constant ka for the inhibition of trypsin by rAAS27.
Fig 6.
Anti-trypsin activity and native AAS27 were observed in A. americanum tick saliva.
Tick saliva was collected from Amblyomma americanum unfed adult females as described in Material and Methods. (A) Tick saliva harvested from unfed ticks was resolved on 12% SDS-PAGE gels and subjected to silver staining (2 μg of total protein resolved) and western blot analysis (1 μg total protein resolved) using a monospecific antibody produced against yeast-expressed rAAS27 (1.0 μg/μL in a 1:500 dilution) and purified total IgG from pre-immune serum (0.50 μg/μL in a 1:250 dilution). (B) Tick saliva (1 μg) was tested against pancreatic bovine trypsin (2 nM). Tick saliva and protease were pre-incubated for 15 min at 37°C in 20 mM Tris-HCl, 150 mM NaCl, Tween 0.01%, pH 7.4 buffer. Substrate was added in a 100 μL final reaction volume and substrate hydrolysis was measured at OD405nm every 15s for 15 min at 30°C. Data presented is representative of three independent replicates and results are plotted as mean of a reading in duplicate. (C) Tick saliva harvested from ticks (1 μg) was incubated with trypsin (0.1 μg) for 1 hour at 37°C. Samples were subjected to SDS-PAGE 12% followed by western blot analysis using anti-rAAS27 monospecific antibody (1.0 μg/μL in a 1:250 dilution).
Fig 7.
rAAS27 binds antibodies to both Amblyomma americanum and Ixodes scapularis tick saliva proteins.
Purified rAAS27 was treated with deglycosylation enzyme mix (D+) or without treatment (D-) and resolved on a 12% SDS-PAGE following western blotting analysis with: (A) rabbit serum generated by repeated A. americanum adult tick infestation (dilution 1:50 and 1:250), (B) rabbit serum generated by repeated adult I. scapularis infestations (dilution 1:50), (C) rabbit serum generated by nymph I. scapularis infestations (dilution 1:50), (D) monospecific antibody produced against yeast-expressed rAAS27 was used as positive control (0.30 μg/μL in a 1:2,000 dilution) and (E) and rabbit pre-immune serum (dilution 1:50).
Fig 8.
Monospecific antibody to rAAS27 dose responsively inhibits its inhibitory activity.
Purified IgG (various doses) from monospecific serum anti-rAAS27 or pre-immune (PI) serum were incubated with rAAS27 (68 nM at 37°C for 30 min before the addition of trypsin (1.5 nM) following an additional 15-min incubation at 37°C. Subsequently, the trypsin substrate was added and protease kinetics was monitored for 15 min every 15 s. Data is reported as residual enzymatic activity.
Fig 9.
rAAS27 blocks formalin- and compound 48/80-induced acute inflammation in vivo.
(A) Rat paw edema induced by intradermal injection of 100 μL of formalin (0.03%) in saline (circles), or co-injected with 25 μg of rAAS27 (squares), or 100 μL of rAAS27 only (triangles). (B) Rat paw edema induced by intradermal injection of 100 μL of compound 48/80 (1 μg) in saline (circles), or co-injected with 25 μg of rAAS27 (squares), or 100 μL of rAAS27 only (triangles). Edema formation was estimated using a digital pletismometer before injection of the agonist at different intervals. Posterior paws from 5 animals were used for each data point. Unpaired t-test was used for statics analysis and p ≤0.05 was considered statistically significant.
Fig 10.
rAAS27 reduces formalin-induced vascular permeability in vivo.
Vascular permeability (Miles assay) was performed by intravenous injection of 700 μL Evan’s blue dye into the tails of rats. Ten minutes later, 100 μL of (i) saline, (ii) formalin 0.3% only, (iii) formalin 0.3% co-injected with rAAS27 (25 μg), or (iv) only 25°μg rAAS27 was injected intradermally on the back of anhestized animals. Sixty minutes after injecting the Evans blue dye, animals were sacrificed and skin removed and spots at injection sites were photographed. The extravasated Evans blue dye was estimated after extraction with formamide and reading at 620 nm. Results are the average of experiments obtained with 6 animals (02 spots per animal). *P = 0.0054 (t-test). Unpaired t-test was used for statics analysis and p ≤0.05 was considered statistically significant.