Purification of N-terminal Chimeras of CCL19 and CCL21 for CCR7 Biased Signaling Analysis Pontian E. Adogamhe 1and Christopher T. Veldkamp1, 2 1University of Wisconsin-Whitewater; 2Medical College of Wisconsin. Research Approach Protein Production Abstract Chemokines are a family of signaling proteins that traffic immune cells throughout the body during homeostatic processes or to areas of infection or inflammation. The chemokines CCL19 and CCL21 are secreted by tissues in the lymphatic system and recruit dendritic cells and naïve T-cells expressing the chemokine receptor CCR7 to the lymph nodes. In addition, when cancer cells express CCR7, CCL19 and CCL21 can contribute to the recruitment these metastatic cancer cells have on the lymph nodes resulting in metastases. CCR7 exhibits ligand bias, eliciting different cellular responses upon activation with CCL19 or CCL21. CCL19 and CCL21 both contain a conserved chemokine domain made up of approximately the first 70-80 amino acids. We hypothesize the N-terminal residues of CCL19 and CCL21 upon binding cause CCR7 to have distinct conformations that results in the observed biased signaling. To test this hypothesis, recombinant N-terminal CCL19 and CCL21 chimeric proteins will be expressed and purified. 1 111 Collection of insoluble His6-SUMO-Chimera 1 or 2 by centrifugation Recombinant expression of a His6-SUMO-Chimera 1 or 2 fusion protein in E. coli followed by cell lysis through sonication Native CCL21 1 77 Native CCL19 SDS-PAGE Analysis of Nickel (II) Affinity Chromatography Solubilization using denaturing conditions and nickel (II) affinity chromatography to isolate His6-SUMO-Chimera 1 or 2 Chimera 1 Flow through & washes E. coli proteins 1 16 111 CCL19 CCL21 Chimera 2 Elution 1 16 77 His6 SUMO Chimera 1 or 2 CCL21 CCL19 Dialysis/refolding SDS-PAGE Analysis of SUMO Protease Digestion His6 SUMO Chimera 1 or 2 Figure 4. Proposed N-terminal CCL19 and CCL21 chimeric proteins. Native CCL21 contains 111 amino acids while native CCL19 contains 77 residues. The extended C-terminus of CCL21 contributes to glycosaminoglycan binding. In order to test the hypothesis that the N-termini of CCL19 and CCL21 result in the differential or biased CCR7 signaling, we propose producing two chimeric proteins. In chimera 1, the first 16 amino acids of CCL19 will replace the first 16 amino acids of CCL21. In chimera 2, the first 16 amino acids of CCL21 will replace the first 16 amino acids of CCL19. Introduction SUMO protease digestion His6-SUMO-CCL19 1-16 CCL21 17-111 Blood vessel or lymphatic vessel lumen His6 SUMO Chimera 1 or 2 His6-SUMO CCL19 1-16 CCL21 17-111 Chemokine receptor SDS-PAGE Analysis of Cation Exchange Chromatography Chemokine concentration gradient Cation Exchange Chromatography Chimera 1 Chimera 2 CCL19 N-terminus/CCL21 CCL21 N-terminus/CCL19 SDF1 Flow through & washes His6 SUMO Cells secreting chemokine Elution Figure 1. A schematic of the process of chemokine controlled cellular chemotaxis. Adapted from reference1. Chemokines are small secreted proteins produced at the site of infection or for homeostatic purposes in order to recruit immune cells to the site of chemokine production. Migrating immune cells express chemokine receptors and move or chemotax towards higher chemokine concentrations. His6-SUMO CCL19 1-16 CCL21 17-111 Chimera 1 or 2 Retained CCL19 signaling response? Retained CCL21 signaling response? Reverse phase HPLC Site 1 Site 2 Membrane Figure 5. Do the proposed N-terminal CCL19 and CCL21 chimeric proteins activate CCR7 similarly to the wild-type chemokine with the same N-terminal amino acid sequence? Adapted from reference3. In chimera 1, the first 16 amino acids of CCL19 will replace the first 16 amino acids of CCL21, and we propose to determine if this chimera signals similarly to CCL19, CCL21 or uniquely. In chimera 2, the first 16 amino acids of CCL21 will replace the first 16 amino acids of CCL19, and we propose to determine if this chimera signals similarly to CCL19, CCL21 or uniquely. Such experiments will provide valuable information regarding which part of the chemokines is responsible for the differential or biased signaling. This information may be helpful in designing drugs that target one type of signaling through CCR7 versus another. Intracellular Figure 6. Flow chart for the purification methods for the N-terminal chimeras with data for the purification of the CCL19 1-16 CCL21 17-111 chimera shown. Figure 2. Chemokines activate chemokine receptors through a two-site two-state binding and activation process. Adapted from reference2. First the extracellular chemokine receptor N-terminus binds to the chemokine, site 1. Then the chemokine N-terminus binds to a second site on the receptor leading to receptor activation and, ultimately, cell migration or chemotaxis towards higher chemokine concentrations. Figure 3. Biased or differential signaling of CCR7 in response to CCL19 and CCL21. Adapted from reference3. CCL19 and CCL21 elicit different cellular signaling and cellular responses upon activation of the CCR7 receptors. An increase in cell signaling or cellular response is indicated by the thickness of the arrow. For example CCL19 leads to greater cell migration or chemotaxis compared to CCL21. Acknowledgements: This material is based upon work supported by the National Science Foundation under Grant No. 1400815. Any opinions, findings, and conclusions or recommendations expressed in this material are those of the author(s) and do not necessarily reflect the views of the National Science Foundation. The Ronald E. McNair baccalaureate program. References: 1. Janeway, C. et al. (2016) Immunobiology 9th ed. New York, NY. Garland Science Publishing. 2. Baggiolini, M. (2001) Chemokines in pathology and medicine, J Intern Med 250, 91-104. 3.  Hjorto, G. M., Larsen, O., Steen, A., Daugvilaite, V., Berg, C., Fares, S., Hansen, M., Ali, S., and Rosenkilde, M. M. (2016) Differential CCR7 Targeting in Dendritic Cells by Three Naturally Occurring CC-Chemokines, Frontiers in immunology 7, 568. 4. Lu, Q., Burns, M. C., McDevitt, P. J., Graham, T. L., Sukman, A. J., Fornwald, J. A., Tang, X., Gallagher, K. T., Hunsberger, G. E., Foley, J. J., Schmidt, D. B., Kerrigan, J. J., Lewis, T. S., Ames, R. S., and Johanson, K. O. (2009) Optimized procedures for producing biologically active chemokines, Protein Expr Purif 62, 251-260. Figure 7. Reverse phase HPLC of the CCL19 1-16 CCL21 17-111 chimera without and with dithiothreitol (DTT) suggest this chimera is oxidized and, therefore, likely folded. The change in retention time without and with DTT suggests the chimera is oxidized, likely through the formation of conserved disulfide bonds found in all chemokines. Future protein NMR experiments will be used to confirm the chimera is folded before the chimera is used by collaborators in the analysis of CCR7 biased signaling.