Citation

  • Authors: Brandum EP. et al.
  • Year: 2022
  • Journal: Int J Mol Sci 23 1397
  • Applications: in vitro / DNA / FectoPRO
  • Cell type: CHO
    Description: Chinese hamster ovary cells

Method

For suspension CHO cells were transfected using FectoPro (200,000 cells/well) in 6-wp. FectoPro transfections were terminated after 3 h by adding FectoPro booster and fresh media. Cells were resuspended in PBS w/glucose and subsequently aliquoted in white 96-well iso plates (~25,000 cells/well). When using C21TP variants, these were added to a final concentration of 10 μM/well, while an equal amount of PBS was added to control cells. The bioluminescence substrate coelenterazine was added to a final conc. of 5 µM. After 10 min, varying ligand concentrations were added. For BRET measurements of Gαi signaling, forskolin was added 5 min after the addition of the ligand to a final conc. of 5 µM. The plates were kept in the dark at all times. A Perkin Almer Envision machine was used for measuring the emission signals at 530 and 480 nm. The BRET signal was determined as the ratio eYFP(530 nm)/Rluc(480 nm).

Abstract

The chemokine receptor CCR7 and its ligands CCL19 and CCL21 regulate the lymph node homing of dendritic cells and naïve T-cells and the following induction of a motile DC-T cell priming state. Although CCL19 and CCL21 bind CCR7 with similar affinities, CCL21 is a weak agonist compared to CCL19. Using a chimeric chemokine, CCL19CCL21N-term|C-term, harboring the N-terminus and the C-terminus of CCL21 attached to the core domain of CCL19, we show that these parts of CCL21 act in a synergistic manner to lower ligand potency and determine the way CCL21 engages with CCR7. We have published that a naturally occurring basic C-terminal fragment of CCL21 (C21TP) boosts the signaling of both CCL19 and CCL21. Boosting occurs as a direct consequence of C21TP binding to the CCR7 N-terminus, which seems to free chemokines with basic C-termini from an unfavorable interaction with negatively charged posttranslational modifications in CCR7. Here, we confirm this using a CCL19-variant lacking the basic C-terminus. This variant displays a 22-fold higher potency at CCR7 compared to WT CCL19 and is highly unaffected by the presence of C21TP. WT CCL19 has a short basic C-terminus, CCL21 a longer one. Here, we propose a way to differentially boost CCL19 and CCL21 activity as short and long versions of C21TP boost CCL19 activity, whereas only a long C21TP version can boost chemokines with a full-length CCL21 C-terminus.

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