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Lookup NU author(s): Professor Akane Kawamura
This work is licensed under a Creative Commons Attribution 4.0 International License (CC BY 4.0).
© 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 < 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.
Author(s): Eaton JRO, Alenazi Y, Singh K, Davies G, Geis-Asteggiante L, Kessler B, Robinson CV, Kawamura A, Bhattacharya S
Publication type: Article
Publication status: Published
Journal: Journal of Biological Chemistry
Print publication date: 20/04/2018
Online publication date: 27/02/2018
Acceptance date: 22/02/2018
Date deposited: 14/10/2019
ISSN (print): 0021-9258
ISSN (electronic): 1083-351X
Publisher: American Society for Biochemistry and Molecular Biology Inc.
PubMed id: 29487134
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