Abstract
Chemokines are a group of chemotaxis proteins that regulate cell trafficking and play important roles in immune responses and inflammation. Ticks are blood-sucking parasites that secrete numerous immune-modulatory agents in their saliva to evade host immune responses. Evasin-3 is a small salivary protein that belongs to a class of chemokine-binding proteins isolated from the brown dog tick, Rhipicephalus sanguineus. Evasin-3 has been shown to have a high affinity for chemokines CXCL1 and CXCL8 and to diminish inflammation in mice. In the present study, solution NMR spectroscopy was used to investigate the structure of Evasin-3 and its CXCL8–Evasin-3 complex. Evasin-3 is found to disrupt the glycosaminoglycan-binding site of CXCL8 and inhibit the interaction of CXCL8 with CXCR2. Structural data were used to design two novel CXCL8-binding peptides. The linear tEv3 17–56 and cyclic tcEv3 16–56 dPG Evasin-3 variants were chemically synthesized by solid-phase peptide synthesis. The affinity of these newly synthesized variants to CXCL8 was measured by surface plasmon resonance biosensor analysis. The Kd values of tEv3 17–56 and tcEv3 16–56 dPG were 27 and 13 nm, respectively. Both compounds effectively inhibited CXCL8-induced migration of polymorphonuclear neutrophils. The present results suggest utility of synthetic Evasin-3 variants as scaffolds for designing and fine-tuning new chemokine-binding agents that suppress immune responses and inflammation.
Highlights
Chemokines are a group of chemotaxis proteins that regulate cell trafficking and play important roles in immune responses and inflammation
Evasins are a family of tick salivary chemokine-binding proteins comprised of two subfamilies
The Cys26–Cys39 bond protrudes through the ring comprised by the Cys22–Cys37 and Cys33–Cys50 forming an inhibitory cystine-knot (ICK) motif
Summary
Chemokines are a group of chemotaxis proteins that regulate cell trafficking and play important roles in immune responses and inflammation. Ticks gain particular interest because their saliva contains numerous bioactive compounds, including proteins, and could be a rich source of candidates for drug development [13] Among those bioactive compounds are Evasins, a class of chemokine-binding proteins first isolated from saliva of the brown dog tick Rhipicephalus sanguineus [14, 15]. Evasin-3 could be considered as a promising alternative to antibodies and chemokine-binding peptides, combining high-affinity and easy accessibility by chemical synthesis because of its relatively small size. We developed two new high-affinity CXCL8-binding peptides based on the experimentally determined structure of the CXCL8 –Evasin-3 complex. Both peptides showed potent anti-inflammatory activity in vitro
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