The N-terminal domain of a tick evasin is critical for chemokine binding and neutralization and confers specific binding activity to other evasins

Journal article

Tick chemokine-binding proteins (evasins) are an emerging class of biologicals that target multiple chemokines and show anti-inflammatory activities in preclinical disease models. Using yeast surface display, we identified a CCL8-binding evasin, P672, from the tick Rhipicephalus pulchellus. We found that P672 binds CCL8 and eight other CC-class chemokines with a Kd < 10 nM and four other CC chemokines with a Kd between 10 and 100 nM and neutralizes CCL3, CCL3L1, and CCL8 with an IC50 < 10 nM. The CC chemokine–binding profile was distinct from that of evasin 1 (EVA1), which does not bind CCL8. We also show that P672's binding activity can be markedly modulated by the location of a StrepII-His purification tag. Combining native MS and bottom-up proteomics, we further demonstrated that P672 is glycosylated and forms a 1:1 complex with CCL8, disrupting CCL8 homodimerization. Homology modeling of P672 using the crystal structure of the EVA1 and CCL3 complex as template suggested that 44 N-terminal residues of P672 form most of the contacts with CCL8. Replacing the 29 N-terminal residues of EVA1 with the 44 N-terminal residues of P672 enabled this hybrid evasin to bind and neutralize CCL8, indicating that the CCL8-binding properties of P672 reside, in part, in its N-terminal residues. This study shows that the function of certain tick evasins can be manipulated simply by adding a tag. We conclude that homology modeling helps identify regions with transportable chemokine-binding functions within evasins, which can be used to construct hybrid evasins with altered properties.

Authors: Eaton, J; Alenazi, Y; Singh, K; Davies, G; Geis-Asteggiante, L

• Publication status: Published
• Peer review status: Peer reviewed
• Publisher: American Society for Biochemistry and Molecular Biology
• Journal: Journal of Biological Chemistry
• Volume: 293
• Issue: 16
• Pages: 6134-6146
• Publication date: 2018-02-27
• Acceptance date: 2018-02-27
• DOI: 10.1074/jbc.ra117.000487
• EISSN: 1083-351X
• ISSN: 0021-9258
• Pmid: 29487134
• Language: English
• Keywords: tick; chemotaxis; evasin; domain swapping; homology modeling; protein-protein interaction; anti-inflammatory therapy; chemokine; inflammation; mass spectrometry (MS)