Purification of a serine protease and evidence for a protein C activator from the saliva of the tick, Ixodes scapularis

Abstract

The saliva of ticks is critical to their survival as parasites and hematophagous animals. In this study, we have purified an enzyme with trypsin-like activity from the saliva of the tick vector of Lyme Disease, Ixodes scapularis. This enzyme, named as IXOSP (I. scapularis salivary serine protease), is a 29.9 kDa molecule with N-terminus FPxMVxLRIKxR. A BLAST search identified IXOSP as a secreted serine protease (AAY66740) with a conserved catalytic triad His, Asp, and Ser. In vitro studies demonstrated that IXOSP cleaves chromogenic substrates with arginine in the P1 position, by a mechanism inhibited by PMSF or aprotinin. Gene expression studies revealed that IXOSP is expressed at different tick developmental stages, including eggs, and unfed or fed adult tick salivary glands, but not in nymphs or in the midgut. While the physiological substrate for IXOSP remains to be identified, we demonstrated that I. scapularis saliva activate protein C (PC) resulting in the production of activated PC, a potent anticoagulant that also regulates a myriad of inflammatory responses through protease activated receptors. In contrast, the salivary glands of Anopheles gambiae, Anopheles stephensi, Anopheles albimanus, Aedes aegypti, Lutzomyia longipalpis, and Phlebotomus ariasi did not activate protein C. These discoveries are discussed in the context of blood coagulation, inflammation and vector-host interactions.

Keywords: Activated protein C (APC); Ixodes scapularis; Protease activated receptors (PAR); Saliva; Serine protease.

Fig. 1. Purification of IXOSP

(A) Saliva was applied to benzamidine-affinity column chromatography, eluted, and tested for amidolytic activity. Active fractions were applied to a HPLC-Reverse C18 column using a gradient from 10% to 80% acetonitrile, formic acid 0.1% for 40 min at a flow rate of 10 μl/min. The eluate was monitored at 225 nm. All fractions were concentrated in a speed-vac and checked for amidolytic activity. (B) MALDI/TOF MS analysis of purified IXOSP. Spectra were analyzed to the most intense matrix peak at m/z 29960.7. (C) SDS-PAGE. Purified IXOSP was loaded in a 12.5% SDS-PAGE. Gels were Coomassie brilliant blue stained. Lanes: 1, Prestained molecular weight marker; 2, HPLC purified protein (peak 2).

Fig. 2. cDNA of IXOSP

(A) The full length clone for AY 66740 is depicted. The N-teminus is underlined. The putative activation peptide is boxed, and the N-terminus identified by Edman degradation is italicized. The catalytic triad His (H), Asp (D) and Ser (S) is circled. (B) Zymogram of purified IXOSP. Purified IXOSP was loaded in a 12.5% SDS-PAGE containing 3.0mg/ml of gelatin. Following electrophoresis, the gels were washed with 2% Triton-X-100 to remove SDS and incubated at 37°C in 100 mM Tris-HCl, pH 8.0. Gels were Commassie Blue stained. Lanes: 1, Prestained molecular weight marker; 2, HPLC purified protein (peak 2) from Figure 1A.

Fig. 3. Gene expression of IXOSP

(A) RT-PCR was carried out using gene-specific primers for IXOSP. Total RNA was extracted from tissues from different developmental stages of ticks. All experiments were carried out in triplicate. cDNA from different tick tissues were PCR amplified using gene-specific primers fro (A) IXOSP or (B) β-actin. The tissues used for RNA extraction are indicated in the figure.

Fig. 4. I. scapularis saliva activates protein C

(A) Saliva (1 μl) of I. scapularis or SGH (1 pair/assay) from different mosquitoes were incubated with PC (100 nM) overnight in EBM-2 media (100 μl, total volume), followed by addition of substrate specific for PC, S2366 (250 μM). Substrate hydrolysis was detected at 405 nm. (B) Saliva (0.5 μl) was incubated with PC in Hepes saline, 0.3 % BSA overnight, in the presence of CaCl₂ (10 mM), ZnCl₂ (100 μM) and compared to activation carried out in the presence of EBM-2 containing 0.3% BSA. In some experiments EDTA was added to chelate divalent ions. (C) Saliva (200 μl) was loaded in a G-75 chromatography column, and eluted with PBS. Twenty μl of each fraction was incubate with 100 nM PC overnight in EBM-2-BSA, and S2366 hydrolysis was estimated at 405 nm (squares). As a control, 20 μl of saliva was incubated with S2366, in the absence of PC (up triangle).