Inflammation is body’s physiological response to any injury or infection. A major feature of inflammatory cascade is the recruitment of leukocyte which in fact is controlled by chemokines (~50 proteins; divided into two major families – CC and CXC). Chemokines bind to their receptors that are expressed on the surface of leukocytes and mediate their migration towards the site of the affected tissue [1]. Due to the bidirectional promiscuity of chemokine-receptor interaction, targeting chemokines stands as a promising approach of anti-inflammatory therapy. Ticks are blood-sucking arachnids that parasitise mammals up to two weeks. To subvert the host immune system, they secrete an arsenal of chemokine-binding proteins called “Evasins”. Several studies have characterised Evasins and their therapeutic potential against inflammation. The validated Evasins are classified into two broad classes: Class A (including Evasin-1 and Evasin-4) contains conserved eight cysteine residues forming four disulfide bonds and binds to CC chemokines exclusively, whereas Class B (including Evasin-3) contains conserved six cysteines forming 3 disulfide bonds and binds selectively to CXC chemokines [3]. Using Evasin-1 and -4, our group performed sequence similarity searches to identify numerous putative Evasin sequences from various tick species, including EVA-RPU-01 and EVA-RPU-02 from Rhipicephalus pulchellus that have conserved eight cysteine residues [2]. Herein, we have expressed these Evasin candidates using E. coli and mammalian (HEK) cells. We demonstrated chemokine-binding activity of these candidates to several CC chemokines by fluorescence anisotropy assay. The structural basis of chemokine recognition and selectivity of chemokine binding will be studied using various biochemical and biophysical methods.
[1] R. P. Bhusal, S. R. Foster, and M. J. Stone, “Structural basis of chemokine and receptor interactions: Key regulators of leukocyte recruitment in inflammatory responses,” Protein Science. 2019, doi: 10.1002/pro.3744.
[2] J. Hayward et al., “Ticks from diverse genera encode chemokine-inhibitory evasin proteins,” J. Biol. Chem., 2017, doi: 10.1074/jbc.M117.807255.
[3] R. P. Bhusal et al., “Evasins: Tick Salivary Proteins that Inhibit Mammalian Chemokines,” Trends in Biochemical Sciences. 2019, doi: 10.1016/j.tibs.2019.10.003.