Alenazi Y

Bhattacharya S

Davies G

Eaton JRO

Geis-Asteggiante L

Kawamura A

Kessler B

Robinson CV

Singh K

\ua9 2018 by The American Society for Biochemistry and Molecular Biology, Inc.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 &lt; 10 nM and four other CC chemokines with a Kd between 10 and 100 nM and neutralizes CCL3, CCL3L1, and CCL8 with an IC50 &lt; 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

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

https://eprint.ncl.ac.uk/fulltext.aspx?url=260793/2D78CD81-C4F4-4361-A25C-A815F3376072.pdf&pub_id=260793

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

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