Leukocyte recruitment is a central characteristic of inflammation and is mediated by chemokines that bind and activate chemokine receptors expressed on leukocytes1. A hallmark of some inflammatory diseases is the overexpression of chemokines that gives rise to excessive or inappropriate recruitment of leukocytes. As such, inhibition of these chemokines represents a promising approach towards targeted treatment of inflammatory diseases2. Evasins are small proteins that have been shown to bind and inhibit host chemokines to facilitate blood feeding in the Brown Dog Tick3. Using bioinformatics tools, we have identified more than 250 putative evasin sequences from a wide variety of hard tick species4. When assessing the sequences of the new evasin candidates we noted that a large number possessed conserved acidic amino acid stretches that flanked tyrosine residues – a known motif for tyrosine sulfation. We therefore predicted that tyrosine sulfation would likely be a native post-translational modification that may influence the chemokine binding and inhibitory activity. Here we will describe the semi-synthesis of a small library of homogeneously sulfated variants of ACA-01, a novel evasin candidate. The (sulfo)proteins were efficiently prepared via native chemical ligation of bacterially expressed C-terminal fragment with chemically synthesized, differentially sulfated N-terminal thioester peptides. We show unequivocally that sulfation of ACA-01 significantly improves affinity for a selection of pro-inflammatory chemokines as well as inhibition of chemokine receptor signalling in mammalian cells. These results provide an essential knowledge base for future efforts to use modified evasins to target specific groups of chemokines for the treatment of inflammatory disease.
1)Trends Immunol., 2004, 25, 75. 2)Nat. Immunol., 2001, 2, 108. 3) Front. Immunol., 2016, 7, 208. 4) JBC, 2017, 292(38), 15670.