Molecular cloning and characterization of a highly selective chemokine-binding protein from the tick Rhipicephalus sanguineus.
Ticks are blood-feeding parasites that secrete a number of immuno-modulatory factors to evade the host immune response. Saliva isolated from different species of ticks has recently been shown to contain chemokine neutralizing activity. To characterize this activity, we constructed a cDNA library from the salivary glands of the common brown dog tick, Rhipicephalus sanguineus. Pools of cDNA clones from the library were transfected into HEK293 cells, and the conditioned media from the transfected cells were tested for chemokine binding activity by chemical cross-linking to radiolabeled CCL3 followed by SDS-PAGE. By de-convolution of a single positive pool of 270 clones, we identified a full-length cDNA encoding a protein of 114 amino acids, which after signal peptide cleavage was predicted to yield a mature protein of 94 amino acids that we called Evasin-1. Recombinant Evasin-1 was produced in HEK293 cells and in insect cells. Using surface plasmon resonance we were able to show that Evasin-1 was exquisitely selective for 3 CC chemokines, CCL3 and CCL4 and the closely related chemokine CCL18, with K(D) values of 0.16, 0.81, and 3.21 nm, respectively. The affinities for CCL3 and CCL4 were confirmed in competition receptor binding assays. Analysis by size exclusion chromatography demonstrated that Evasin-1 was monomeric and formed a 1:1 complex with CCL3. Thus, unlike the other chemokine-binding proteins identified to date from viruses and from the parasitic worm Schistosoma mansoni, Evasin-1 is highly specific for a subgroup of CC chemokines, which may reflect a specific role for these chemokines in host defense against parasites.