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.
Frauenschuh
Achim
A
Merck Serono Geneva Research Centre, CH-1211 Geneva, Switzerland.
Power
Christine A
CA
Merck Serono Geneva Research Centre, CH-1211 Geneva, Switzerland. Electronic address: christine.power@merckserono.net.
Déruaz
Maud
M
Merck Serono Geneva Research Centre, CH-1211 Geneva, Switzerland.
Ferreira
Beatriz R
BR
Department of Biochemistry and Immunology, School of Medicine of Ribeirão Preto, USP, São Paulo, SP14049-900, Brazil, and.
Silva
João S
JS
Department of Biochemistry and Immunology, School of Medicine of Ribeirão Preto, USP, São Paulo, SP14049-900, Brazil, and.
Teixeira
Mauro M
MM
Departamento de Bioquimica e Imunologia, Instituto de Ciencias Biologicas, Universidade Federal de Minas Gerais, Belo Horizonte, MG, B1270-901, Brazil.
Dias
João M
JM
Merck Serono Geneva Research Centre, CH-1211 Geneva, Switzerland.
Martin
Thierry
T
Merck Serono Geneva Research Centre, CH-1211 Geneva, Switzerland.
Wells
Timothy N C
TNC
Merck Serono Geneva Research Centre, CH-1211 Geneva, Switzerland.
Proudfoot
Amanda E I
AEI
Merck Serono Geneva Research Centre, CH-1211 Geneva, Switzerland. Electronic address: amanda.proudfoot@merckserono.net.
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D054405:Chemokine CCL3; D054407:Chemokine CCL4; D018925:Chemokines / Q000378:metabolism*; D019742:Chemokines, CC / Q000378:metabolism; D003001:Cloning, Molecular; D004285:Dogs; D005260:Female; D006801:Humans; D008969:Molecular Sequence Data; D019707:Receptors, Chemokine / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D048508:Rhipicephalus sanguineus / Q000737:chemistry*; D012469:Salivary Glands / Q000737:chemistry; D020349:Surface Plasmon Resonance
C515429:CCL3 protein, human; C066507:CCL4 protein, human; D002352:Carrier Proteins; D054405:Chemokine CCL3; D054407:Chemokine CCL4; D018925:Chemokines; D019742:Chemokines, CC; C523601:Evasin-1, Rhipicephalus sanguineus; D019707:Receptors, Chemokine; D011994:Recombinant Proteins